WO2023056328A2 - Solid supports and methods for depleting and/or enriching library fragments prepared from biosamples - Google Patents
Solid supports and methods for depleting and/or enriching library fragments prepared from biosamples Download PDFInfo
- Publication number
- WO2023056328A2 WO2023056328A2 PCT/US2022/077221 US2022077221W WO2023056328A2 WO 2023056328 A2 WO2023056328 A2 WO 2023056328A2 US 2022077221 W US2022077221 W US 2022077221W WO 2023056328 A2 WO2023056328 A2 WO 2023056328A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- rna
- library
- sequence
- solid support
- oligonucleotides
- Prior art date
Links
- 239000012634 fragment Substances 0.000 title claims abstract description 447
- 238000000034 method Methods 0.000 title claims abstract description 376
- 239000007787 solid Substances 0.000 title claims abstract description 264
- 230000000779 depleting effect Effects 0.000 title claims abstract description 112
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims abstract description 439
- 239000000523 sample Substances 0.000 claims abstract description 323
- 108020004418 ribosomal RNA Proteins 0.000 claims abstract description 152
- 238000012163 sequencing technique Methods 0.000 claims abstract description 74
- 108091034117 Oligonucleotide Proteins 0.000 claims description 344
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 claims description 214
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 130
- 230000000295 complement effect Effects 0.000 claims description 115
- 239000000203 mixture Substances 0.000 claims description 97
- 239000002299 complementary DNA Substances 0.000 claims description 95
- 244000005700 microbiome Species 0.000 claims description 90
- 108020004414 DNA Proteins 0.000 claims description 83
- 150000007523 nucleic acids Chemical group 0.000 claims description 79
- 239000003298 DNA probe Substances 0.000 claims description 72
- 108020003215 DNA Probes Proteins 0.000 claims description 55
- 230000027455 binding Effects 0.000 claims description 43
- 230000003321 amplification Effects 0.000 claims description 38
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 38
- 102000006382 Ribonucleases Human genes 0.000 claims description 37
- 108010083644 Ribonucleases Proteins 0.000 claims description 37
- 108020004999 messenger RNA Proteins 0.000 claims description 29
- 230000001580 bacterial effect Effects 0.000 claims description 21
- 230000000593 degrading effect Effects 0.000 claims description 21
- 239000011324 bead Substances 0.000 claims description 16
- 102000004190 Enzymes Human genes 0.000 claims description 11
- 108090000790 Enzymes Proteins 0.000 claims description 11
- 230000002441 reversible effect Effects 0.000 claims description 10
- 102000016911 Deoxyribonucleases Human genes 0.000 claims description 8
- 108010053770 Deoxyribonucleases Proteins 0.000 claims description 8
- 108060003196 globin Proteins 0.000 claims description 8
- 102000018146 globin Human genes 0.000 claims description 8
- 238000000746 purification Methods 0.000 claims description 7
- 238000011161 development Methods 0.000 claims description 6
- 108010008532 Deoxyribonuclease I Proteins 0.000 claims description 5
- 102000007260 Deoxyribonuclease I Human genes 0.000 claims description 5
- 102100038614 Hemoglobin subunit gamma-1 Human genes 0.000 claims description 5
- 102100038617 Hemoglobin subunit gamma-2 Human genes 0.000 claims description 5
- 101001031977 Homo sapiens Hemoglobin subunit gamma-1 Proteins 0.000 claims description 5
- 101001031961 Homo sapiens Hemoglobin subunit gamma-2 Proteins 0.000 claims description 5
- 244000005702 human microbiome Species 0.000 abstract description 46
- 230000002255 enzymatic effect Effects 0.000 abstract description 9
- 102000053602 DNA Human genes 0.000 description 71
- 102000039446 nucleic acids Human genes 0.000 description 68
- 108020004707 nucleic acids Proteins 0.000 description 68
- 238000002360 preparation method Methods 0.000 description 52
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 40
- 241000894007 species Species 0.000 description 27
- 239000002773 nucleotide Substances 0.000 description 22
- 125000003729 nucleotide group Chemical group 0.000 description 22
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical group [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 21
- 230000000813 microbial effect Effects 0.000 description 19
- 102100034343 Integrase Human genes 0.000 description 18
- 241000894006 Bacteria Species 0.000 description 17
- 101710203526 Integrase Proteins 0.000 description 17
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 15
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 15
- 230000000368 destabilizing effect Effects 0.000 description 15
- 238000009396 hybridization Methods 0.000 description 15
- 239000000126 substance Substances 0.000 description 15
- 238000006243 chemical reaction Methods 0.000 description 13
- 239000002699 waste material Substances 0.000 description 12
- 239000013614 RNA sample Substances 0.000 description 11
- 244000005709 gut microbiome Species 0.000 description 11
- 238000003559 RNA-seq method Methods 0.000 description 10
- 239000003795 chemical substances by application Substances 0.000 description 10
- 206010028980 Neoplasm Diseases 0.000 description 9
- 241000588724 Escherichia coli Species 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
- 235000014469 Bacillus subtilis Nutrition 0.000 description 7
- 101001028702 Homo sapiens Mitochondrial-derived peptide MOTS-c Proteins 0.000 description 7
- 102100037173 Mitochondrial-derived peptide MOTS-c Human genes 0.000 description 7
- 238000012408 PCR amplification Methods 0.000 description 7
- 230000008901 benefit Effects 0.000 description 7
- 239000000872 buffer Substances 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 7
- 238000010804 cDNA synthesis Methods 0.000 description 6
- 230000015556 catabolic process Effects 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 238000003776 cleavage reaction Methods 0.000 description 6
- 238000006731 degradation reaction Methods 0.000 description 6
- 238000013461 design Methods 0.000 description 6
- 230000006870 function Effects 0.000 description 6
- 229920000642 polymer Polymers 0.000 description 6
- 230000007017 scission Effects 0.000 description 6
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 description 5
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 5
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 5
- AHCYMLUZIRLXAA-SHYZEUOFSA-N Deoxyuridine 5'-triphosphate Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C[C@@H]1N1C(=O)NC(=O)C=C1 AHCYMLUZIRLXAA-SHYZEUOFSA-N 0.000 description 5
- 241000736262 Microbiota Species 0.000 description 5
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 description 5
- 229960003237 betaine Drugs 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 238000003908 quality control method Methods 0.000 description 5
- 230000002829 reductive effect Effects 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 241000203069 Archaea Species 0.000 description 4
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 4
- 238000013459 approach Methods 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 201000011510 cancer Diseases 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 230000007613 environmental effect Effects 0.000 description 4
- 238000013467 fragmentation Methods 0.000 description 4
- 238000006062 fragmentation reaction Methods 0.000 description 4
- 230000014509 gene expression Effects 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 238000011002 quantification Methods 0.000 description 4
- 244000005714 skin microbiome Species 0.000 description 4
- 239000002689 soil Substances 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 108020004465 16S ribosomal RNA Proteins 0.000 description 3
- 108020005096 28S Ribosomal RNA Proteins 0.000 description 3
- 108091093088 Amplicon Proteins 0.000 description 3
- 241000186016 Bifidobacterium bifidum Species 0.000 description 3
- 108091028075 Circular RNA Proteins 0.000 description 3
- 108020004635 Complementary DNA Proteins 0.000 description 3
- 241000233866 Fungi Species 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- 238000002123 RNA extraction Methods 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 238000003491 array Methods 0.000 description 3
- 230000004888 barrier function Effects 0.000 description 3
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 3
- 230000006862 enzymatic digestion Effects 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 238000003384 imaging method Methods 0.000 description 3
- 230000003100 immobilizing effect Effects 0.000 description 3
- 238000013507 mapping Methods 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 238000002844 melting Methods 0.000 description 3
- 230000008018 melting Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 102000040430 polynucleotide Human genes 0.000 description 3
- 108091033319 polynucleotide Proteins 0.000 description 3
- 239000002157 polynucleotide Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- -1 1,5-anhydrohexitol nucleic acid Chemical class 0.000 description 2
- 241000186000 Bifidobacterium Species 0.000 description 2
- 241000193403 Clostridium Species 0.000 description 2
- 108010092160 Dactinomycin Proteins 0.000 description 2
- 241001608234 Faecalibacterium Species 0.000 description 2
- 241000207202 Gardnerella Species 0.000 description 2
- 208000022559 Inflammatory bowel disease Diseases 0.000 description 2
- 241001112693 Lachnospiraceae Species 0.000 description 2
- 241000186660 Lactobacillus Species 0.000 description 2
- 241000927544 Olsenella Species 0.000 description 2
- 108091093037 Peptide nucleic acid Proteins 0.000 description 2
- 241000605861 Prevotella Species 0.000 description 2
- 241001453443 Rothia <bacteria> Species 0.000 description 2
- 241000194017 Streptococcus Species 0.000 description 2
- 108091046915 Threose nucleic acid Proteins 0.000 description 2
- 241001148134 Veillonella Species 0.000 description 2
- 229930183665 actinomycin Natural products 0.000 description 2
- 230000000692 anti-sense effect Effects 0.000 description 2
- 229940002008 bifidobacterium bifidum Drugs 0.000 description 2
- 238000005842 biochemical reaction Methods 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 2
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 2
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 2
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000012938 design process Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000010195 expression analysis Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 239000010931 gold Substances 0.000 description 2
- 229910052737 gold Inorganic materials 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000010348 incorporation Methods 0.000 description 2
- 229940039696 lactobacillus Drugs 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 238000007481 next generation sequencing Methods 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 239000013610 patient sample Substances 0.000 description 2
- 238000010791 quenching Methods 0.000 description 2
- 244000000000 soil microbiome Species 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 230000009870 specific binding Effects 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 230000002103 transcriptional effect Effects 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 229940035893 uracil Drugs 0.000 description 2
- 239000002351 wastewater Substances 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- MPCAJMNYNOGXPB-UHFFFAOYSA-N 1,5-Anhydro-mannit Natural products OCC1OCC(O)C(O)C1O MPCAJMNYNOGXPB-UHFFFAOYSA-N 0.000 description 1
- PHIYHIOQVWTXII-UHFFFAOYSA-N 3-amino-1-phenylpropan-1-ol Chemical compound NCCC(O)C1=CC=CC=C1 PHIYHIOQVWTXII-UHFFFAOYSA-N 0.000 description 1
- 108020004513 Bacterial RNA Proteins 0.000 description 1
- 241001202853 Blautia Species 0.000 description 1
- 108091033409 CRISPR Proteins 0.000 description 1
- 238000010354 CRISPR gene editing Methods 0.000 description 1
- 102000004594 DNA Polymerase I Human genes 0.000 description 1
- 108010017826 DNA Polymerase I Proteins 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108700039887 Essential Genes Proteins 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 108060002716 Exonuclease Proteins 0.000 description 1
- 108091093094 Glycol nucleic acid Proteins 0.000 description 1
- 108700005443 Microbial Genes Proteins 0.000 description 1
- 206010061309 Neoplasm progression Diseases 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 1
- 102000015623 Polynucleotide Adenylyltransferase Human genes 0.000 description 1
- 108010024055 Polynucleotide adenylyltransferase Proteins 0.000 description 1
- 108010026552 Proteome Proteins 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 238000012167 Small RNA sequencing Methods 0.000 description 1
- 108091046869 Telomeric non-coding RNA Proteins 0.000 description 1
- 239000012805 animal sample Substances 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- AIYUHDOJVYHVIT-UHFFFAOYSA-M caesium chloride Chemical compound [Cl-].[Cs+] AIYUHDOJVYHVIT-UHFFFAOYSA-M 0.000 description 1
- 230000023852 carbohydrate metabolic process Effects 0.000 description 1
- 235000021256 carbohydrate metabolism Nutrition 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- HGCIXCUEYOPUTN-UHFFFAOYSA-N cis-cyclohexene Natural products C1CCC=CC1 HGCIXCUEYOPUTN-UHFFFAOYSA-N 0.000 description 1
- 230000001010 compromised effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 230000036267 drug metabolism Effects 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 102000013165 exonuclease Human genes 0.000 description 1
- 230000002550 fecal effect Effects 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 125000001153 fluoro group Chemical group F* 0.000 description 1
- 238000007672 fourth generation sequencing Methods 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 238000011331 genomic analysis Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 208000028774 intestinal disease Diseases 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000012804 iterative process Methods 0.000 description 1
- 238000010208 microarray analysis Methods 0.000 description 1
- 108091027963 non-coding RNA Proteins 0.000 description 1
- 102000042567 non-coding RNA Human genes 0.000 description 1
- 239000002853 nucleic acid probe Substances 0.000 description 1
- 238000001821 nucleic acid purification Methods 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- 238000005580 one pot reaction Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 230000037452 priming Effects 0.000 description 1
- 230000000135 prohibitive effect Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000007841 sequencing by ligation Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 230000005751 tumor progression Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
-
- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B40/00—Libraries per se, e.g. arrays, mixtures
- C40B40/04—Libraries containing only organic compounds
- C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
- C40B40/08—Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
-
- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
- C40B50/14—Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
Definitions
- This disclosure relates to solid supports and methods for depleting library fragments prepared from unwanted RNA sequences and/or enriching library fragments prepared from desired RNA sequences. Libraries enriched or depleted with the present methods may be used to generate sequencing data. Also described are probes and methods for enzymatic depletion of ribosomal RNA from human microbiome samples.
- RNA typically comprises most of the RNA molecules in total RNA (approximately 80%-95%).
- rRNA ribosomal RNA
- rRNAs ribosomal RNA
- mRNAs globin messenger RNAs
- RNA-Seq sequencing RNA transcripts
- RNase H-mediated depletion involves hybridizing DNA probes complementary to known rRNA sequences followed by DNA:RNA hybrid-specific cleavage by RNase H and subsequent removal via wash steps.
- This methodology is implemented as part of the current Illumina Total RNA Stranded Library Prep workflow and New England Biolabs NEBNext rRNA Depletion Kit and RNA depletion methods as described in US Patent Nos. 9,745,570 and 9,005,891. While these methods are effective, drawbacks include upfront depletion, increased costs, and increased hands-on time (HOT).
- multi-omic data incorporate measurements of the microbiota-associated transcriptome, proteome, or metabolome providing further insights into microbiome activity and function.
- metagenomic and metatranscriptomic profiles tend to be generally consistent, microbial functional profiles derived from DNA sequencing are more conserved across donors than transcriptional profiles, which are highly donor specific (Franzosa, E. A. et al. Proc. Natl. Acad. Sci. U. S. A. ll l(22):E2329-38 (2014)).
- many broadly encoded metagenomic pathways are expressed by a small number of organisms, highlighting the utility of metatranscriptomics to identify functional activities (Abu- Ah, G. S. et al. Nat.
- rRNA microbial ribosomal RNA
- RNA ribosomal RNA
- rRNA ribosomal RNA
- non- ribosomal RNA can be easily and efficiently enriched through selective reverse transcription or pull-down approaches that target the poly-A tail or using probes to specifically bind rRNA molecules prior to removal by capture or enzymatic digestion (Hrdlickova et al. Wiley Interdiscip. Rev. RNA 8(l):10.1002/wma.l364 (2017) and Zhao et al. Sci. Rep.
- composition of the microbiota varies substantially across body sites and throughout different life stages, further expanding the taxonomic coverage required for robust depletion of rRNA across human microbiome samples.
- Probe-based sequence capture methods such as were employed with Illumina’s RiboZero Gold kit can provide strong rRNA depletion across a variety of sample types, including human gut microbiome samples (Reck, M. et al. BMC Genomics 16(1): 494 (2015)).
- probes are costly, difficult to manufacture, and tend to perform best with high quality RNA samples.
- capture-based rRNA depletion methods can yield variable results based on operator skill. These factors led to the discontinuation of the capture based bacterial RiboZero Gold depletion kit.
- Described herein is the development of a pan-human microbiome probe set for efficient and consistent enzymatic (RNase H) microbial rRNA depletion.
- RNase H enzymatic microbial rRNA depletion
- probes were designed that effectively deplete rRNA found in human oral, vaginal and adult and infant gut microbiome samples, substantially improving mapping rates to coding microbial gene databases.
- the rRNA depletion process was shown to not introduce substantial bias in the metatranscriptomic profiles.
- the resulting metatranscriptomics data allows the user to refine informatic pipelines for rRNA and host mapping and to examine gene expression and functional pathways across human microbiome sites.
- the method described here circumvents the limitations of sequence capture methods and represents a highly effective rRNA depletion option for metatranscriptomics studies of human-associated microbial communities.
- rRNA depletion is often performed by using hybridization with 16S and 23S rRNA probes followed by separation or by using depletion of rRNAs using a method based on binding of probes following by exonuclease treatment. After rRNA depletion, library preparation can be performed.
- Described herein is an iterative probe design strategy that was used to develop a probe set for efficient enzymatic rRNA removal of human-associated microbiota. This strategy resulted in custom probe sets that efficiently deplete rRNA from a range of human microbiome samples, including adult gut, infant gut, oral, and vaginal communities. Successful rRNA depletion allows for characterization of taxonomic and functional changes during the development of the gut microbiome. Further, the rRNA depletion process does not introduce substantial quantitative error in the resulting transcriptomic profiles.
- the panhuman microbiome enzymatic rRNA depletion probes described here provide a powerful tool for studying the transcriptional dynamics and function of the human microbiome.
- methods of “upfront depletion,” including RNase depletion, can be problematic for users with limited total RNA material for input into the assay. For example, if insufficient RNA remains after upfront depletion methods, downstream biochemical reactions can be inefficient resulting in poor assay performance and results. Further, upfront depletion with RNase H includes wash steps (potentially causing loss of desired RNA) and high temperature incubations (potentially causing degradation of desired RNA), which may be a concern with certain samples.
- Described herein is a differentiated solution using a solid support (such as a flowcell-like device) with immobilized oligonucleotides that can bind to library fragments prepared from unwanted RNA.
- a solid support such as a flowcell-like device
- immobilized oligonucleotides that can bind to library fragments prepared from unwanted RNA.
- library fragments prepared from rRNA sequences can be captured by flowcell-tethered oligonucleotides, while library fragments lacking these sequences can be siphoned for collection.
- After collection of the non-depleted library fragments only a quick quality control step checking the concentration and size of the non-rRNA sequencing library may be performed prior to standard sequencing. This approach is advantageous as rRNA can act as a “carrier molecule” for low abundance RNA molecules throughout the library preparation process, making for a robust, sensitive assay.
- this method can be expanded to substitute traditional PCR amplification via thermal cycler in favor of a bridge amplification-like process to further reduce HOT and demonstrate additional library preparation functionality via sequencer fluidics chemistry. Similar methods can also be used for other unwanted RNA, such as for depleting host-derived RNA transcripts when a user wants to specifically evaluate microbiome RNA from a host.
- RNA depleting methods prepared from unwanted RNA and methods of enriching library fragments prepared from desired RNA. These methods may be performed with standard lab equipment, such as flowcells comprised in sequencers.
- standard sequencing consumables and platform i.e., sequencer
- depletion or enrichment is performed after cDNA synthesis and amplification.
- probes that may be used for enzymatic depletion of rRNA from human microbiome samples.
- Embodiment 1 A method of depleting unwanted cDNA library fragments from a library of cDNA fragments prepared from RNA, wherein the unwanted library fragments comprise those prepared from unwanted RNA sequences, comprising (a) preparing a solid support comprising at least one immobilized oligonucleotide, wherein each immobilized oligonucleotide comprises a nucleic acid sequence corresponding to an unwanted RNA sequence or its complement, (b) adding the library of fragments to the solid support and hybridizing the library fragments to at least one immobilized oligonucleotide to allow binding of unwanted library fragments to at least one immobilized oligonucleotide, and (c) collecting library fragments not bound to at least one immobilized oligonucleotide.
- Embodiment 2 The method of embodiment 1, wherein at least one unwanted RNA sequence has at least 90%, at least 95%, or at least 99% homology to a high-abundance RNA sequence in a sample used to prepare the library of fragments.
- Embodiment 3 The method of embodiment 2, wherein all unwanted sequences have at least 90%, at least 95%, or at least 99% homology to a high-abundance RNA sequence in a sample used to prepare the library of fragments.
- Embodiment 4 The method of embodiment 1, wherein the at least one unwanted RNA sequence is a high-abundance RNA sequence.
- Embodiment 5 The method of any one of embodiments 2-4, wherein the high- abundance RNA sequence is a ribosomal RNA (rRNA) sequence.
- rRNA ribosomal RNA
- Embodiment 6 The method of any one of embodiments 1-5, wherein the unwanted RNA sequence is comprised in a host transcriptome.
- Embodiment 7 The method of any one of embodiments 1-6, wherein the unwanted RNA sequence is globin mRNA or 28S, 23S, 18S, 5.8S, 5S, 16S, 12S, HBA-A1, HBA-A2, HBB, HBB-B1, HBB-B2, HBG1, or HBG2 RNA, or a fragment thereof.
- Embodiment 8 The method of any one of embodiments 1-7, wherein the unwanted RNA sequence is from human, rat, mouse, or bacteria.
- Embodiment 9 The method of embodiment 8, wherein the unwanted RNA sequence is 28S, 18S, 5.8S, 5S, 16S, or 12S RNA from humans, or a fragment thereof.
- Embodiment 10 The method of embodiment 8, wherein the unwanted RNA sequence is rat 16S, rat 28S, mouse 16S, or mouse 28S RNA.
- Embodiment 11 The method of embodiment 8, wherein the bacteria are Archaea species, E. Coli, or B. subtilis.
- Embodiment 12 The method of embodiment 8, wherein the unwanted RNA sequence is comprised in 23S, 16S, or 5S RNA from Gram-positive or Gram-negative bacteria.
- Embodiment 13 The method of embodiment 8, wherein the unwanted RNA sequence is from an organism in the human microbiome.
- Embodiment 14 The method of embodiment 13, wherein the at least one immobilized oligonucleotide comprises a sequence comprising any one or more of SEQ ID NOs: 1-1131 or its complement.
- Embodiment 15 The method of any one of embodiments 1-14, wherein the at least one immobilized oligonucleotide comprises 2 or more, 5 or more, 10 or more, 25 or more, 50 or more, 100 or more, 200 or more, 300 or more, 400 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, 1000 or more, 1100 or more, or 1131 sequences selected from SEQ ID NOs: 1-1131 or its complement.
- Embodiment 16 The method of embodiment 15, wherein the at least one immobilized oligonucleotide comprises 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, 1000 or more, 1100 or more, or 1131 sequences selected from SEQ ID NOs: 1- 1131 or its complement.
- Embodiment 17 The method of any one of embodiments 14-16, wherein the at least one immobilized oligonucleotide comprises at least one sequence comprising at least one of SEQ ID NOs: 1-10, 12-18, 21, 22, 24-33, 35, 39-43, 45-48, 50-73, 75, 77, 78, 81-84, 86-103, 105-107, 109-127, 129-134, 136-140, 142, 143, 148, 149, 151-155, 157, 160-165, 168-174, 176-182, 184-187, 189-194, 196-204, 206, 208-215, 217-223, 225, 227-246, 248- 265, 269, 270, 272-277, 279, 281, 282, 284-290, 292-301, 303-321, 323-331, 333-336, 338, 340-342, 344, 346-349, 351-355, 357-359, 361-372
- Embodiment 18 The method of embodiment 17, wherein the at least one immobilized oligonucleotide comprises 100 or more, 500 or more, or 1000 or more sequences comprising at least one of SEQ ID NOs: 1-10, 12-18, 21, 22, 24-33, 35, 39-43, 45- 48, 50-73, 75, 77, 78, 81-84, 86-103, 105-107, 109-127, 129-134, 136-140, 142, 143, 148, 149, 151-155, 157, 160-165, 168-174, 176-182, 184-187, 189-194, 196-204, 206, 208-215, 217-223, 225, 227-246, 248-265, 269, 270, 272-277, 279, 281, 282, 284-290, 292-301, 303- 321, 323-331, 333-336, 338, 340-342, 344, 346-349, 351-355, 357-359
- Embodiment 19 The method of embodiment 18, wherein the at least one immobilized oligonucleotide comprises a sequence comprising each of SEQ ID NOs: 1-10, 12-18, 21, 22, 24-33, 35, 39-43, 45-48, 50-73, 75, 77, 78, 81-84, 86-103, 105-107, 109-127, 129-134, 136-140, 142, 143, 148, 149, 151-155, 157, 160-165, 168-174, 176-182, 184-187, 189-194, 196-204, 206, 208-215, 217-223, 225, 227-246, 248-265, 269, 270, 272-277, 279, 281, 282, 284-290, 292-301, 303-321, 323-331, 333-336, 338, 340-342, 344, 346-349, 351- 355, 357-359, 361-372, 374-380, 383
- Embodiment 20 The method of any one of embodiments 17-19, wherein the at least one immobilized oligonucleotide further comprises at least one sequence comprising at least one of SEQ ID NOs: 19, 74, 76, 85, 104, 108, 158, 175, 226, 278, 322, 339, 389, 513, 517, 520, 546, 553, 609, 611, 650, 662, 677, 683, 686, 706, 780, 827, 835, 882, 1022, 1059, 1077, 1078, 1098, 1106, 1111, 1114, 1128, and 1131 or its complement.
- Embodiment 21 The method of embodiment 20, wherein the pool of oligonucleotides comprises 10 or more, 20 or more, or 30 or more sequences comprising at least one of SEQ ID NOs: 19, 74, 76, 85, 104, 108, 158, 175, 226, 278, 322, 339, 389, 513, 517, 520, 546, 553, 609, 611, 650, 662, 677, 683, 686, 706, 780, 827, 835, 882, 1022, 1059, 1077, 1078, 1098, 1106, 1111, 1114, 1128, and 1131 or its complement.
- Embodiment 22 The method of embodiment 21, wherein the pool of oligonucleotides comprises a sequence comprising each of SEQ ID NOs: 19, 74, 76, 85, 104, 108, 158, 175, 226, 278, 322, 339, 389, 513, 517, 520, 546, 553, 609, 611, 650, 662, 677, 683, 686, 706, 780, 827, 835, 882, 1022, 1059, 1077, 1078, 1098, 1106, 1111, 1114, 1128, and 1131 or its complement.
- Embodiment 23 The method of any one of embodiments 14-16, wherein the pool of oligonucleotides comprises at least one sequence comprising at least one of SEQ ID NOs: 1-10, 12-19, 21, 22, 24-33, 35, 39-43, 45-48, 50-78, 81-127, 129-134, 136-140, 142, 143, 148, 149, 151-155, 157, 158, 160-165, 168-182, 184-187, 189-194, 196-204, 206, 208- 215, 217-223, 225-246, 248-265, 269, 270, 272-279, 281, 282, 284-290, 292-301, 303-331, 333-336, 338-342, 344, 346-349, 351-355, 357-359, 361-372, 374-380, 383-386, 388-391, 393, 395-401, 403-406, 408-420, 422-439, 441, 443, 4
- Embodiment 24 The method of embodiment 23, wherein the pool of oligonucleotides comprises 100 or more, 500 or more, or 1000 or more sequences comprising at least one of SEQ ID NOs: 1-10, 12-19, 21, 22, 24-33, 35, 39-43, 45-48, 50-78, 81-127, 129-134, 136-140, 142, 143, 148, 149, 151-155, 157, 158, 160-165, 168-182, 184-187, 189- 194, 196-204, 206, 208-215, 217-223, 225-246, 248-265, 269, 270, 272-279, 281, 282, 284- 290, 292-301, 303-331, 333-336, 338-342, 344, 346-349, 351-355, 357-359, 361-372, 374- 380, 383-386, 388-391, 393, 395-401, 403-406, 408-420, 422-439
- Embodiment 25 The method of embodiment 24, wherein the at least one immobilized oligonucleotide comprises a sequence comprising each of SEQ ID NOs: 1-10, 12-19, 21, 22, 24-33, 35, 39-43, 45-48, 50-78, 81-127, 129-134, 136-140, 142, 143, 148, 149, 151-155, 157, 158, 160-165, 168-182, 184-187, 189-194, 196-204, 206, 208-215, 217-223, 225-246, 248-265, 269, 270, 272-279, 281, 282, 284-290, 292-301, 303-331, 333-336, 338- 342, 344, 346-349, 351-355, 357-359, 361-372, 374-380, 383-386, 388-391, 393, 395-401, 403-406, 408-420, 422-439, 441, 443, 444, 446-460,
- Embodiment 26 The method of any one of embodiments 17-25, wherein the at least one immobilized oligonucleotide further comprises at least one sequence comprising at least one of SEQ ID NOs: 11, 20, 23, 34, 36-38, 44, 49, 79, 80, 128, 135, 141, 144-147, 150, 156, 159, 166, 167, 183, 188, 195, 205, 207, 216, 224, 247, 266-268, 271, 280, 283, 291, 302, 332, 337, 343, 345, 350, 356, 360, 373, 381, 382, 387, 392, 394, 402, 407, 421, 440,
- Embodiment 27 The method of embodiment 26, wherein the at least one immobilized oligonucleotide comprises 10 or more, 20 or more, or 30 or more sequences comprising at least one of SEQ ID NOs: 11, 20, 23, 34, 36-38, 44, 49, 79, 80, 128, 135, 141, 144-147, 150, 156, 159, 166, 167, 183, 188, 195, 205, 207, 216, 224, 247, 266-268, 271, 280, 283, 291, 302, 332, 337, 343, 345, 350, 356, 360, 373, 381, 382, 387, 392, 394, 402, 407,
- Embodiment 28 The method of embodiment 27, wherein the at least one immobilized oligonucleotide comprises a sequence comprising each of SEQ ID NOs: 11, 20, 23, 34, 36-38, 44, 49, 79, 80, 128, 135, 141, 144-147, 150, 156, 159, 166, 167, 183, 188, 195, 205, 207, 216, 224, 247, 266-268, 271, 280, 283, 291, 302, 332, 337, 343, 345, 350, 356, 360, 373, 381, 382, 387, 392, 394, 402, 407, 421, 440, 442, 445, 461, 467, 470, 472, 478, 503,
- Embodiment 29 The method of any one of embodiments 1-28, wherein the unwanted RNA sequences are selected by determining the most abundant sequences in a sample comprising RNA.
- Embodiment 30 The method of embodiment 29, wherein the most abundant sequences comprise the 100 most abundant sequences, the 1,000 most abundant sequences, or the 10,000 most abundant sequences.
- Embodiment 31 The method of any one of embodiments 1-30, wherein the unwanted RNA sequence comprises a sequence with homology of at least 90%, at least 95%, or at least 99% to a most abundant sequence in a sample comprising RNA.
- Embodiment 32 The method of any one of embodiments 1-31, wherein the collected library fragments comprise a library depleted of unwanted library fragments.
- Embodiment 33 The method of any one of embodiments 32, wherein unwanted library fragments serve as carrier molecules for other library fragments.
- Embodiment 34 The method of any one of embodiments 1-33, wherein the library of fragments added to the solid support is prepared from RNA using a stranded method of cDNA preparation.
- Embodiment 35 The method of any one of embodiments 1-34, wherein the library fragments comprise library adapters and the solid support further comprises immobilized oligonucleotides comprising solid support adapter sequences that can bind to library adapters.
- Embodiment 36 The method of embodiment 35, wherein the solid support adapter sequences comprise a P5 sequence (SEQ ID NO: 1132), a P7 sequence (SEQ ID NO: 1133), and/or their complements.
- Embodiment 37 The method of embodiment 35 or embodiment 36, wherein adapter complements that are all or partially complementary to the solid support adapter sequences are bound to the solid support adapter sequences.
- Embodiment 38 The method of embodiment 37, wherein the binding of the adapter complements to the solid support adapter sequences is reversible.
- Embodiment 39 The method of embodiment 37 or embodiment 38, wherein adapter complements bound to the solid support adapter sequences generate double-stranded immobilized oligonucleotides.
- Embodiment 40 The method of embodiment 39, wherein solid support adapter sequences bound to adapter complements cannot bind to library adapters.
- Embodiment 41 The method of embodiment 39 or embodiment 40, further comprising denaturing library fragments and/or adapter complements hybridized to the immobilized oligonucleotides.
- Embodiment 42 The method of embodiment 41, wherein the denaturing is performed with a denaturing agent and/or heat.
- Embodiment 43 The method of embodiment 42, wherein the denaturing agent is NaOH, optionally wherein the NaOH concentration is 0.2 N.
- Embodiment 44 The method of embodiment 42, wherein the heat is 95°C - 98°C.
- Embodiment 45 The method of any one of embodiments 41-44, wherein the denatured library fragments and/or adapter complements are siphoned to a waste compartment.
- Embodiment 46 The method of any one of embodiments 41-45, wherein the steps of adding a sample, collecting, and denaturing are repeated, wherein the collected library fragments are added back to the solid support after the denaturing.
- Embodiment 47 The method of any one of embodiments 1-46, wherein the collected library fragments are collected in a reservoir comprised in a sequencer comprising the flowcell.
- Embodiment 48 The method of any one of embodiments 1-47, wherein the library fragments that hybridize to immobilized oligonucleotides comprise library fragments prepared from rRNA.
- Embodiment 49 The method of any one of embodiments 1-48, wherein the library depleted of unwanted library fragments comprises fewer library fragments prepared from unwanted RNA sequences, as compared to the same library before it was added to the solid support.
- Embodiment 50 The method of any one of embodiments 1-49, wherein the unwanted library fragments that hybridize to immobilized oligonucleotides comprise library fragments prepared from host RNA comprised in a sample comprising host RNA and nonhost nucleic RNA.
- Embodiment 51 The method of embodiment 50, wherein the non-host RNA is microbial.
- Embodiment 52 The method of embodiment 51, wherein microbe is a bacterium, a virus, and/or a fungus.
- Embodiment 53 The method of embodiment 52, wherein the microbe is a pathogen.
- Embodiment 54 The method of embodiment 52, wherein the microbe is an organism in the host microbiome.
- Embodiment 55 The method of any one of embodiments 50-54, wherein the host is human.
- Embodiment 56 The method of any one of embodiments 29-55, further comprising adding the collected library fragments to the solid support after denaturing the hybridized library fragments and/or adapter complements.
- Embodiment 57 The method of embodiment 1-56, wherein sequences comprised in library fragments specifically bind to solid support adapter sequences comprising P5 (SEQ ID NO: 1132), P7 (SEQ ID NO: 1133), and/or their complements.
- Embodiment 58 The method of embodiment 1-57, wherein library adapter sequences are added to collected library fragments.
- Embodiment 59 The method of embodiment 58, wherein the library adapter sequences are added by ligation.
- Embodiment 60 The method of any one of embodiments 1-59, wherein the library of fragments added to the solid support is prepared by a method comprising incorporating one or more library adapters that specifically bind to solid support adapter sequences comprising P5 (SEQ ID NO: 1132), P7 (SEQ ID NO: 1133), and/or their complements.
- Embodiment 61 The method of embodiment 60, wherein the method comprising incorporating one or more library adapters is tagmentation or fragmentation followed by adapter ligation.
- Embodiment 62 The method of any one of embodiments 1-61, wherein the method does not require degradation of RNA.
- Embodiment 63 The method of any one of embodiments 1-62, wherein the library depleted of unwanted library fragments is assessed for library size and/or concentration.
- Embodiment 64 The method of any one of embodiments 1-63, wherein the library depleted of unwanted library fragments is sequenced.
- Embodiment 65 The method of any one of embodiments 1-64, further comprising amplifying the library depleted of unwanted library fragments before sequencing.
- Embodiment 66 The method of embodiment 65, wherein the amplifying is by PCR amplification.
- Embodiment 67 The method of embodiment 65, wherein the amplifying is by bridge amplification.
- Embodiment 68 The method of embodiment 67, wherein bridge amplification is performed after adding the collected library fragments to the solid support and allowing the library adapters comprised in the collected library fragments to bind to the solid support adapter sequences, wherein the adding is performed after denaturing the hybridized library fragments and/or adapter complements.
- Embodiment 69 The method of embodiment 64, 65, 67, or 68, wherein the sequencing is performed without PCR amplification.
- Embodiment 70 The method of any one of embodiments 64, 65, or 67-69, wherein the amplifying does not require a thermocycler.
- Embodiment 71 The method of any one of embodiments 1-70, wherein the method is fully performed in a sequencer.
- Embodiment 72 A method of enriching desired cDNA library fragments from a library of cDNA fragments prepared from RNA, wherein the desired library fragments comprise those prepared from desired RNA sequences, comprising (a) preparing a solid support comprising at least one immobilized oligonucleotide, wherein each immobilized oligonucleotide comprises a nucleic acid sequence corresponding to a desired RNA sequence or its complement, (b) adding the library of fragments to the solid support and hybridizing the library fragments to the at least one immobilized oligonucleotide to allow binding of desired library fragments to the at least one immobilized oligonucleotide, and (c) collecting library fragments bound to the at least one immobilized oligonucleotide.
- Embodiment 73 The method of embodiment 72, wherein the library of fragments has been subjected to a method of depleting unwanted cDNA library fragments of any one of embodiments 1-71 before the adding.
- Embodiment 74 The method of embodiment 72 or 73, wherein at least one desired RNA sequence has at least 90%, at least 95%, or at least 99% homology to an RNA sequence of interest in a sample used to prepare the library of fragments.
- Embodiment 75 The method of embodiment 74, wherein all desired RNA sequences have at least 90%, at least 95%, or at least 99% homology to an RNA sequence of interest in a sample used to prepare the library of fragments.
- Embodiment 76 The method of any one of embodiments 72-75, wherein the at least one desired RNA sequence is an RNA sequence of interest.
- Embodiment 77 The method of any one of embodiments 72-76, wherein the desired RNA sequence is an exome sequence.
- Embodiment 78 The method of any one of embodiments 72-77, wherein the desired RNA sequence is from human, rat, mouse, and/or bacteria.
- Embodiment 79 The method of embodiment 78, wherein the desired RNA sequence is from an organism in the human microbiome.
- Embodiment 80 The method of any one of embodiments 72-79, wherein the collected library fragments comprise a library enriched for desired library fragments.
- Embodiment 81 The method of any one of embodiments 72-90, wherein the library of fragments added to the solid support is prepared from RNA using a stranded method of cDNA preparation.
- Embodiment 82 The method of any one of embodiments 72-81, wherein the collecting comprises denaturing the library fragments hybridized to the at least one immobilized oligonucleotide and then collecting the library enriched for desired fragments in a reservoir comprised in a sequencer comprising the solid support.
- Embodiment 83 The method of embodiment 82, wherein the denaturing is performed with a denaturing agent and/or heat.
- Embodiment 84 The method of embodiment 83, wherein the heat is 95°C - 98°C.
- Embodiment 85 The method of embodiment 83, wherein the denaturing agent is NaOH, optionally wherein the NaOH concentration is 0.2 N.
- Embodiment 86 The method of any one of embodiments 82-85, wherein the steps of adding the library, denaturing, and collecting are repeated, wherein the collected library fragments are added to the solid support after the denaturing.
- Embodiment 87 The method of any one of embodiments 82-86, wherein the library enriched for desired library fragments comprises a greater percentage of library fragments prepared from desired RNA sequences, as compared to the library before adding to the solid support.
- Embodiment 88 The method of any one of embodiments 82-87, wherein the library enriched for desired library fragments is assessed for library size and/or concentration.
- Embodiment 89 The method of any one of embodiments 82-88, wherein the library enriched for desired library fragments is sequenced.
- Embodiment 90 The method of any one of embodiments 82-89, further comprising amplifying the library enriched for desired library fragments before sequencing.
- Embodiment 91 The method of any one of embodiments 1-90, wherein the at least one immobilized oligonucleotide is 20-100 bases in length, optionally wherein the at least one immobilized oligonucleotides is 45-55 bases in length.
- Embodiment 92 The method of any one of embodiments 1-91, wherein the at least one immobilized oligonucleotide is single-stranded.
- Embodiment 93 The method of any one of embodiments 1-92, wherein single-stranded library fragments are prepared before adding the library of fragments to the solid support.
- Embodiment 94 The method of any one of embodiments 1-93, wherein the solid support is a flowcell.
- Embodiment 95 A solid support having two pools of immobilized oligonucleotides on its surface, wherein the first pool comprises immobilized oligonucleotides each comprising a nucleic acid sequence corresponding to an unwanted RNA sequence or its complement and the second pool comprises immobilized oligonucleotides each comprising a solid support adapter sequence that can bind to a library adapter comprised in library fragments.
- Embodiment 96 The solid support of embodiment 95, wherein at least one unwanted RNA sequence has at least 90%, at least 95%, or at least 99% homology to a high-abundance RNA sequence in a sample used to prepare the library of fragments.
- Embodiment 97 The solid support of embodiment 96, wherein all unwanted sequences have at least 90%, at least 95%, or at least 99% homology to a high- abundance RNA sequence in a sample used to prepare the library of fragments.
- Embodiment 98 The solid support of any one of embodiments 95-97, wherein the at least one unwanted RNA sequence is a high-abundance RNA sequence.
- Embodiment 99 The solid support of any one of embodiments 96-98, wherein the high-abundance RNA sequence is a ribosomal RNA (rRNA) sequence.
- rRNA ribosomal RNA
- Embodiment 100 The solid support of any one of embodiments 95-99, wherein the unwanted RNA sequence is comprised in a host transcriptome.
- Embodiment 101 The solid support of any one of embodiments 95-
- RNA sequence is globin mRNA or 28S, 23S, 18S, 5.8S, 5S, 16S, 12S, HBA-A1, HBA-A2, HBB, HBB-B1, HBB-B2, HBG1, or HBG2 RNA, or a fragment thereof.
- Embodiment 102 The solid support of any one of embodiments 95-
- RNA sequence is from human, rat, mouse, or bacteria.
- Embodiment 103 The solid support of embodiment 102, wherein the unwanted RNA sequence is 28S, 18S, 5.8S, 5S, 16S, or 12S RNA from humans, or a fragment thereof.
- Embodiment 104 The solid support of embodiment 102, wherein the unwanted RNA sequence is comprised in rat 16S, rat 28S, mouse 16S, or mouse 28S RNA.
- Embodiment 105 The solid support of embodiment 102, wherein the bacteria are Archaea species, E. Coli, or B. subtilis.
- Embodiment 106 The solid support of embodiment 102, wherein the unwanted RNA sequence is comprised in 23S, 16S, or 5S RNA from Gram-positive or Gramnegative bacteria.
- Embodiment 107 The solid support of embodiment 102, wherein the unwanted RNA sequence is from an organism comprised in the human microbiome.
- Embodiment 108 The solid support of any one of embodiments 95-
- the unwanted RNA sequence comprises any one or more of SEQ ID NOs: 1- 1131.
- Embodiment 109 The solid support of any one of embodiments 95-
- the unwanted RNA sequence comprises a sequence with homology of at least 90%, at least 95%, or at least 99% to a most abundant sequence in a sample comprising RNA, wherein the most abundant sequences comprise the 100 most abundant sequences, the 1,000 most abundant sequences, or the 10,000 most abundant sequences.
- Embodiment 110 The solid support of any one of embodiments 95-
- solid support adapter sequences comprise a P5 sequence (SEQ ID NO: 1132), a P7 sequence (SEQ ID NO: 1133), and/or their complements.
- Embodiment 111 The solid support of any one of embodiments 95-
- Embodiment 112 The solid support of embodiment 111, wherein the binding of the adapter complements to the solid support adapter sequences is reversible.
- Embodiment 113 The solid support of embodiment 111 or embodiment 112, wherein the solid support adapter sequences and adapter complements generate double-stranded immobilized oligonucleotides.
- Embodiment 114 The solid support of any one of embodiments 95-
- the at least one immobilized oligonucleotide is 20-100 bases in length, optionally wherein the at least one immobilized oligonucleotide is 45-55 bases in length.
- Embodiment 115 The solid support of any one of embodiments 95-
- Embodiment 116 The solid support of any one of embodiments 95-
- Embodiment 117 A composition comprising a single-stranded library fragment comprising cDNA prepared from a sample comprising RNA that is hybridized to the solid support of any one of embodiments 95-116.
- Embodiment 118 The composition of embodiment 117, wherein the cDNA is complementary to RNA comprised in the sample.
- Embodiment 119 A method for depleting unwanted RNA molecules comprised in a patient microbiome sample, wherein the patient microbiome sample comprises at least one target RNA or DNA sequence and at least one unwanted RNA molecule, comprising (a) sequencing a plurality of probe-development microbiome samples to determine at least one unwanted RNA molecule comprising a bacterial ribosomal RNA (rRNA) sequence from sequencing data; (b) preparing a probe set comprising at least one DNA probe complementary to the at least one unwanted RNA molecule; (c) contacting the patient microbiome sample with the probe set to prepare DNA: RNA hybrids; and (d) contacting the DNA:RNA hybrids with a ribonuclease that degrades the RNA from the DNA:RNA hybrids, thereby degrading the unwanted RNA molecules in the patient microbiome sample to form a degraded mixture.
- rRNA bacterial ribosomal RNA
- Embodiment 120 A method for depleting unwanted RNA molecules comprised in a patient microbiome sample, wherein the patient microbiome sample comprises at least one target RNA or DNA sequence and at least one unwanted RNA molecule, comprising (a) contacting the patient microbiome sample with a probe set comprising at least one sequence comprising at least one of SEQ ID NOs: 1-1131 to prepare DNA:RNA hybrids; and
- Embodiment 121 The method of embodiment 119 or embodiment 120, further comprising (a) degrading any remaining DNA probes by contacting the degraded mixture with a DNA digesting enzyme, optionally wherein the DNA digesting enzyme is DNase I, to form a DNA degraded mixture; and (b) separating the degraded RNA from the degraded mixture or the DNA degraded mixture.
- a DNA digesting enzyme optionally wherein the DNA digesting enzyme is DNase I
- Embodiment 122 The method of any one of embodiments 119-121, wherein the contacting with the probe set comprises treating the nucleic acid sample with a destabilizer.
- Embodiment 123 The method of embodiment 122, wherein the destabilizer is heat and/or a nucleic acid destabilizing chemical.
- Embodiment 124 The method of embodiment 123, wherein the nucleic acid destabilizing chemical comprises betaine, DMSO, formamide, glycerol, or a derivative thereof, or a mixture thereof.
- Embodiment 125 The method of embodiment 124, wherein the nucleic acid destabilizing chemical comprises formamide.
- Embodiment 126 The method of embodiment 125, wherein the formamide is present during the contacting with the probe set at a concentration of from about 10 to 45% by volume.
- Embodiment 127 The method of any one of embodiments 123-126, wherein treating the sample with heat comprises applying heat above the melting temperature of the at least one DNA: RNA hybrid.
- Embodiment 128 The method of any one of embodiments 119-127, wherein the ribonuclease is RNase H or hybridase.
- Embodiment 129 The method of any one of embodiments 119-128, wherein the patient is human.
- Embodiment 130 The method of any one of embodiments 119-129, wherein the microbiome sample is oral, vaginal, or from the gut.
- Embodiment 131 The method of embodiment 119-130, wherein the sample from the gut is a stool sample.
- Embodiment 132 The method of embodiment 131, wherein the oral sample is a sample from the tongue.
- Embodiment 133 The method of any one of embodiments 119-132, wherein the at least one DNA probe comprise 2 or more, 5 or more, 10 or more, 25 or more, 50 or more, 100 or more, 200 or more, 300 or more, 400 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, 1000 or more, 1100 or more, or 1131 sequences selected from SEQ ID NOs: 1-1131 or its complement.
- Embodiment 134 The method of embodiment 133, wherein the at least one DNA probe comprises 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, 1000 or more, 1100 or more, or 1131 sequences selected from SEQ ID NOs: 1-1131 or its complement.
- Embodiment 135. The method of any one of embodiments 119-134, wherein the patient is at least 12 months of age, at least 15 months of age, at least 24 months of age, or at least 36 months of age.
- Embodiment 136 The method of any one of embodiments 119-135, wherein the microbiome sample comprises at least one unwanted RNA molecule from Faecalibacterium, Lachnospiraceae, and/or Clostridium.
- Embodiment 137 The method of any one of embodiments 119-136, wherein the microbiome sample is vaginal and comprises at least one unwanted RNA molecule from Gardnerella, Lactobacillus and/or Olsenella.
- Embodiment 138 The method of any one of embodiments 119-136, wherein the microbiome sample is from tongue and comprises at least one unwanted RNA molecule from Veillonella, Rothia, Streptococcus, and/or Prevotella.
- Embodiment 139 The method of any one of embodiments 120-138, wherein the at least one DNA probe comprises at least one sequence comprising at least one of SEQ ID NOs: 1-10, 12-18, 21, 22, 24-33, 35, 39-43, 45-48, 50-73, 75, 77, 78, 81-84, 86- 103, 105-107, 109-127, 129-134, 136-140, 142, 143, 148, 149, 151-155, 157, 160-165, 168- 174, 176-182, 184-187, 189-194, 196-204, 206, 208-215, 217-223, 225, 227-246, 248-265, 269, 270, 272-277, 279, 281, 282, 284-290, 292-301, 303-321, 323-331, 333-336, 338, 340- 342, 344, 346-349, 351-355, 357-359, 361-372, 374-
- Embodiment 140 The method of embodiment 139, wherein the at least one DNA probe comprises 100 or more, 500 or more, or 1000 or more sequences comprising at least one of SEQ ID NOs: 1-10, 12-18, 21, 22, 24-33, 35, 39-43, 45-48, 50-73, 75, 77, 78, 81-84, 86-103, 105-107, 109-127, 129-134, 136-140, 142, 143, 148, 149, 151-155, 157, 160- 165, 168-174, 176-182, 184-187, 189-194, 196-204, 206, 208-215, 217-223, 225, 227-246, 248-265, 269, 270, 272-277, 279, 281, 282, 284-290, 292-301, 303-321, 323-331, 333-336, 338, 340-342, 344, 346-349, 351-355, 357-359, 361-372,
- Embodiment 14E The method of embodiment 140, wherein the at least one DNA probe comprises a sequence comprising each of SEQ ID NOs: 1-10, 12-18, 21, 22, 24-33, 35, 39-43, 45-48, 50-73, 75, 77, 78, 81-84, 86-103, 105-107, 109-127, 129-134, 136- 140, 142, 143, 148, 149, 151-155, 157, 160-165, 168-174, 176-182, 184-187, 189-194, 196- 204, 206, 208-215, 217-223, 225, 227-246, 248-265, 269, 270, 272-277, 279, 281, 282, 284- 290, 292-301, 303-321, 323-331, 333-336, 338, 340-342, 344, 346-349, 351-355, 357-359, 361-372, 374-380, 383-386, 388,
- Embodiment 142 The method of any one of embodiments 139-141, wherein the patient is 3 months of age or younger, 6 months of age of younger, 12 months of age or younger, 18 months of age or younger, 24 months of age or younger, or 36 months of age or younger.
- Embodiment 143 The method of embodiment 142, wherein the microbiome sample comprises at least one unwanted RNA molecules from Bifidobacterium bifidum and/or Blautia.
- Embodiment 144 The method of any one of embodiments 139-143, wherein the at least one DNA probe further comprises at least one sequence comprising at least one of SEQ ID NOs: 19, 74, 76, 85, 104, 108, 158, 175, 226, 278, 322, 339, 389, 513, 517, 520, 546, 553, 609, 611, 650, 662, 677, 683, 686, 706, 780, 827, 835, 882, 1022, 1059, 1077, 1078, 1098, 1106, 1111, 1114, 1128, and 1131.
- Embodiment 145 The method of embodiment 144, wherein the at least one DNA probe comprises 10 or more, 20 or more, or 30 or more sequences comprising at least one of SEQ ID NOs: 19, 74, 76, 85, 104, 108, 158, 175, 226, 278, 322, 339, 389, 513, 517, 520, 546, 553, 609, 611, 650, 662, 677, 683, 686, 706, 780, 827, 835, 882, 1022, 1059, 1077, 1078, 1098, 1106, 1111, 1114, 1128, and 1131.
- Embodiment 146 The method of embodiment 145, wherein the at least one DNA probe comprises a sequence comprising each of SEQ ID NOs: 19, 74, 76, 85, 104, 108, 158, 175, 226, 278, 322, 339, 389, 513, 517, 520, 546, 553, 609, 611, 650, 662, 677,
- Embodiment 147 The method of any one of embodiments 120-138, wherein the at least one immobilized oligonucleotide comprises at least one sequence comprising at least one of SEQ ID NOs: 1-10, 12-19, 21, 22, 24-33, 35, 39-43, 45-48, 50-78, 81-127, 129-134, 136-140, 142, 143, 148, 149, 151-155, 157, 158, 160-165, 168-182, 184- 187, 189-194, 196-204, 206, 208-215, 217-223, 225-246, 248-265, 269, 270, 272-279, 281, 282, 284-290, 292-301, 303-331, 333-336, 338-342, 344, 346-349, 351-355, 357-359, 361- 372, 374-380, 383-386, 388-391, 393, 395-401, 403-406, 408-420, 422-4
- Embodiment 148 The method of embodiment 147, wherein the at least one immobilized oligonucleotide comprises 100 or more, 500 or more, or 1000 or more sequences comprising at least one of SEQ ID NOs: 1-10, 12-19, 21, 22, 24-33, 35, 39-43, 45- 48, 50-78, 81-127, 129-134, 136-140, 142, 143, 148, 149, 151-155, 157, 158, 160-165, 168- 182, 184-187, 189-194, 196-204, 206, 208-215, 217-223, 225-246, 248-265, 269, 270, 272- 279, 281, 282, 284-290, 292-301, 303-331, 333-336, 338-342, 344, 346-349, 351-355, 357- 359, 361-372, 374-380, 383-386, 388-391, 393, 395-401, 403-406, 4
- Embodiment 149 The method of embodiment 148, wherein the at least one immobilized oligonucleotide comprises a sequence comprising each of SEQ ID NOs: 1- 10, 12-19, 21, 22, 24-33, 35, 39-43, 45-48, 50-78, 81-127, 129-134, 136-140, 142, 143, 148, 149, 151-155, 157, 158, 160-165, 168-182, 184-187, 189-194, 196-204, 206, 208-215, 217- 223, 225-246, 248-265, 269, 270, 272-279, 281, 282, 284-290, 292-301, 303-331, 333-336, 338-342, 344, 346-349, 351-355, 357-359, 361-372, 374-380, 383-386, 388-391, 393, 395- 401, 403-406, 408-420, 422-439, 441, 443, 444,
- Embodiment 150 The method of any one of embodiments 139-149, wherein the at least one DNA probe further comprises at least one sequence comprising at least one of SEQ ID NOs: 11, 20, 23, 34, 36-38, 44, 49, 79, 80, 128, 135, 141, 144-147, 150, 156, 159, 166, 167, 183, 188, 195, 205, 207, 216, 224, 247, 266-268, 271, 280, 283, 291, 302, 332, 337, 343, 345, 350, 356, 360, 373, 381, 382, 387, 392, 394, 402, 407, 421, 440, 442,
- Embodiment 151 The method of embodiment 150, wherein the at least one DNA probe comprises 10 or more, 20 or more, or 30 or more sequences comprising at least one of SEQ ID NOs: 11, 20, 23, 34, 36-38, 44, 49, 79, 80, 128, 135, 141, 144-147, 150, 156, 159, 166, 167, 183, 188, 195, 205, 207, 216, 224, 247, 266-268, 271, 280, 283, 291, 302, 332, 337, 343, 345, 350, 356, 360, 373, 381, 382, 387, 392, 394, 402, 407, 421, 440, 442,
- Embodiment 152 The method of embodiment 151, wherein the at least one DNA probe comprises a sequence comprising each of SEQ ID NOs: 11, 20, 23, 34, 36- 38, 44, 49, 79, 80, 128, 135, 141, 144-147, 150, 156, 159, 166, 167, 183, 188, 195, 205, 207, 216, 224, 247, 266-268, 271, 280, 283, 291, 302, 332, 337, 343, 345, 350, 356, 360, 373, 381, 382, 387, 392, 394, 402, 407, 421, 440, 442, 445, 461, 467, 470, 472, 478, 503, 515, 525,
- Embodiment 153 The method of any one of embodiments 119-152, wherein the method depletes 70% or greater, 80% or greater, 90% or greater, or 95% or greater of bacterial rRNA comprised in the microbiome sample.
- Embodiment 154 A composition comprising a probe set comprising
- Embodiment 155 The composition of embodiment 154, wherein the ribonuclease is RNase H.
- Embodiment 156 A kit comprising a probe set comprising (a) at least one DNA probe comprising at least one sequence comprising at least one of SEQ ID NOs: 1- 1131; and (b) a ribonuclease capable of degrading RNA in an DNA:RNA hybrid.
- Embodiment 157 The kit of embodiment 156, comprising (a) a probe set comprising at least one DNA probe comprising at least one of SEQ ID NOs: 1-1131; (b) a ribonuclease; (c) a DNase; and (d) RNA purification beads.
- Embodiment 158 The kit of embodiment 157, wherein the ribonuclease is RNase H.
- Embodiment 159 The kit of embodiment 157 or 158, further comprising an RNA depletion buffer, a probe depletion buffer, and a probe removal buffer.
- Embodiment 160 The kit of any one of embodiments 157-160, further comprising a nucleic acid destabilizing chemical.
- Embodiment 161 The kit of embodiment 160, wherein the nucleic acid destabilizing chemical comprises betaine, DMSO, formamide, glycerol, or a derivative thereof, or a mixture thereof.
- Embodiment 162 The kit of embodiment 161, wherein the nucleic acid destabilizing chemical comprises formamide.
- Embodiment 163 The composition or kit of any one of embodiments 154-162, wherein the at least one DNA probe comprise 2 or more, 5 or more, 10 or more, 25 or more, 50 or more, 100 or more, 200 or more, 300 or more, 400 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, 1000 or more, 1100 or more, or 1131 sequences selected from SEQ ID NOs: 1-1131.
- Embodiment 164 The composition or kit of embodiment 163, wherein the at least one DNA probe comprises 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, 1000 or more, 1100 or more, or 1131 sequences selected from SEQ ID NOs: 1- 1131.
- Embodiment 165 The composition or kit of any one of embodiments 154-164, wherein the at least one DNA probe comprises at least one sequence comprising at least one of SEQ ID NOs: 1-10, 12-18, 21, 22, 24-33, 35, 39-43, 45-48, 50-73, 75, 77, 78, 81- 84, 86-103, 105-107, 109-127, 129-134, 136-140, 142, 143, 148, 149, 151-155, 157, 160-165, 168-174, 176-182, 184-187, 189-194, 196-204, 206, 208-215, 217-223, 225, 227-246, 248- 265, 269, 270, 272-277, 279, 281, 282, 284-290, 292-301, 303-321, 323-331, 333-336, 338, 340-342, 344, 346-349, 351-355, 357-359, 361-372, 3
- Embodiment 166 The composition or kit of embodiment 165, wherein the at least one DNA probe comprises 100 or more, 500 or more, or 1000 or more sequences comprising at least one of SEQ ID NOs: 1-10, 12-18, 21, 22, 24-33, 35, 39-43, 45-48, 50-73, 75, 77, 78, 81-84, 86-103, 105-107, 109-127, 129-134, 136-140, 142, 143, 148, 149, 151-155, 157, 160-165, 168-174, 176-182, 184-187, 189-194, 196-204, 206, 208-215, 217-223, 225, 227-246, 248-265, 269, 270, 272-277, 279, 281, 282, 284-290, 292-301, 303-321, 323-331, 333-336, 338, 340-342, 344, 346-349, 351-355, 357-359, 361-3
- Embodiment 167 The composition or kit of embodiment 166, wherein the at least one DNA probe comprises a sequence comprising each of SEQ ID NOs: 1-10, 12- 18, 21, 22, 24-33, 35, 39-43, 45-48, 50-73, 75, 77, 78, 81-84, 86-103, 105-107, 109-127, 129- 134, 136-140, 142, 143, 148, 149, 151-155, 157, 160-165, 168-174, 176-182, 184-187, 189- 194, 196-204, 206, 208-215, 217-223, 225, 227-246, 248-265, 269, 270, 272-277, 279, 281, 282, 284-290, 292-301, 303-321, 323-331, 333-336, 338, 340-342, 344, 346-349, 351-355, 357-359, 361-372, 374-380, 383-386,
- Embodiment 168 The composition or kit of any one of embodiments 165-167, wherein the at least one DNA probe further comprises at least one sequence comprising at least one of SEQ ID NOs: 19, 74, 76, 85, 104, 108, 158, 175, 226, 278, 322, 339, 389, 513, 517, 520, 546, 553, 609, 611, 650, 662, 677, 683, 686, 706, 780, 827, 835, 882, 1022, 1059, 1077, 1078, 1098, 1106, 1111, 1114, 1128, and 1131.
- Embodiment 169 The composition or kit of embodiment 168, wherein the at least one DNA probe comprises 10 or more, 20 or more, or 30 or more sequences comprising at least one of SEQ ID NOs: 19, 74, 76, 85, 104, 108, 158, 175, 226, 278, 322, 339, 389, 513, 517, 520, 546, 553, 609, 611, 650, 662, 677, 683, 686, 706, 780, 827, 835, 882, 1022, 1059, 1077, 1078, 1098, 1106, 1111, 1114, 1128, and 1131.
- Embodiment 170 The composition or kit of embodiment 169, wherein the at least one DNA probe comprises a sequence comprising each of SEQ ID NOs: 19, 74, 76, 85, 104, 108, 158, 175, 226, 278, 322, 339, 389, 513, 517, 520, 546, 553, 609, 611, 650, 662, 677, 683, 686, 706, 780, 827, 835, 882, 1022, 1059, 1077, 1078, 1098, 1106, 1111, 1114, 1128, and 1131.
- Embodiment 17E The composition or kit of any one of embodiments 154-164, wherein the at least one immobilized oligonucleotide comprises at least one sequence comprising at least one of SEQ ID NOs: 1-10, 12-19, 21, 22, 24-33, 35, 39-43, 45- 48, 50-78, 81-127, 129-134, 136-140, 142, 143, 148, 149, 151-155, 157, 158, 160-165, 168- 182, 184-187, 189-194, 196-204, 206, 208-215, 217-223, 225-246, 248-265, 269, 270, 272- 279, 281, 282, 284-290, 292-301, 303-331, 333-336, 338-342, 344, 346-349, 351-355, 357- 359, 361-372, 374-380, 383-386, 388-391, 393, 395-401, 403-406, 408-420
- Embodiment 172 The composition or kit of embodiment 171, wherein the at least one immobilized oligonucleotide comprises 100 or more, 500 or more, or 1000 or more sequences comprising at least one of SEQ ID NOs: 1-10, 12-19, 21, 22, 24-33, 35, 39- 43, 45-48, 50-78, 81-127, 129-134, 136-140, 142, 143, 148, 149, 151-155, 157, 158, 160-165, 168-182, 184-187, 189-194, 196-204, 206, 208-215, 217-223, 225-246, 248-265, 269, 270, 272-279, 281, 282, 284-290, 292-301, 303-331, 333-336, 338-342, 344, 346-349, 351-355, 357-359, 361-372, 374-380, 383-386, 388-391, 393, 395-401, 403-406, 408-
- Embodiment 173 The composition or kit of embodiment 172, wherein the at least one immobilized oligonucleotide comprises a sequence comprising each of SEQ ID NOs: 1-10, 12-19, 21, 22, 24-33, 35, 39-43, 45-48, 50-78, 81-127, 129-134, 136-140, 142, 143, 148, 149, 151-155, 157, 158, 160-165, 168-182, 184-187, 189-194, 196-204, 206, 208- 215, 217-223, 225-246, 248-265, 269, 270, 272-279, 281, 282, 284-290, 292-301, 303-331, 333-336, 338-342, 344, 346-349, 351-355, 357-359, 361-372, 374-380, 383-386, 388-391, 393, 395-401, 403-406, 408-420, 422-439, 441, 443, 444
- Embodiment 174 The composition or kit of any one of embodiments 165-173, wherein the at least one DNA probe further comprises at least one sequence comprising at least one of SEQ ID NOs: 11, 20, 23, 34, 36-38, 44, 49, 79, 80, 128, 135, 141, 144-147, 150, 156, 159, 166, 167, 183, 188, 195, 205, 207, 216, 224, 247, 266-268, 271, 280, 283, 291, 302, 332, 337, 343, 345, 350, 356, 360, 373, 381, 382, 387, 392, 394, 402, 407,
- Embodiment 175. The composition or kit of embodiment 174, wherein the at least one DNA probe comprises 10 or more, 20 or more, or 30 or more sequences comprising at least one of SEQ ID NOs: 11, 20, 23, 34, 36-38, 44, 49, 79, 80, 128, 135, 141, 144-147, 150, 156, 159, 166, 167, 183, 188, 195, 205, 207, 216, 224, 247, 266-268, 271, 280, 283, 291, 302, 332, 337, 343, 345, 350, 356, 360, 373, 381, 382, 387, 392, 394, 402, 407,
- Embodiment 176 The composition or kit of embodiment 175, wherein the at least one DNA probe comprises a sequence comprising each of SEQ ID NOs: 11, 20, 23, 34, 36-38, 44, 49, 79, 80, 128, 135, 141, 144-147, 150, 156, 159, 166, 167, 183, 188, 195, 205, 207, 216, 224, 247, 266-268, 271, 280, 283, 291, 302, 332, 337, 343, 345, 350, 356, 360, 373, 381, 382, 387, 392, 394, 402, 407, 421, 440, 442, 445, 461, 467, 470, 472, 478, 503,
- Embodiment 177 A method of selecting cDNA library fragments from a library of cDNA fragments prepared from RNA, comprising (a) preparing a solid support comprising a pool of immobilized oligonucleotides, wherein each immobilized oligonucleotide in the pool comprises a nucleic acid sequence corresponding to an RNA sequence or its complement, (b) adding the library of fragments to the solid support and hybridizing the library fragments to at least one immobilized oligonucleotide to allow binding of library fragments to at least one immobilized oligonucleotide, and (c) collecting library fragments either bound or not bound to at least one immobilized oligonucleotide.
- Embodiment 178 The method of embodiment 177, wherein (a) the selecting is depleting unwanted cDNA library fragments, wherein the RNA sequence comprises an unwanted RNA sequence, the unwanted library fragments comprise those prepared from unwanted RNA sequences, and the collecting comprises collecting library fragment not bound to at least one immobilized oligonucleotide; or (b) the selecting is enriching desired cDNA library fragments, wherein the RNA sequence comprises a desired RNA sequence, the desired library fragments comprise those prepared from desired RNA sequences, and the collecting comprises collecting library fragment bound to at least one immobilized oligonucleotide.
- Embodiment 179 Embodiment 179.
- Embodiment 180 A solid support having two pools of immobilized oligonucleotides on its surface, wherein the first pool of oligonucleotides comprises immobilized oligonucleotides each comprising a nucleic acid sequence corresponding to an unwanted RNA sequence or its complement and the second pool of oligonucleotides comprises immobilized oligonucleotides each comprising a solid support adapter sequence that can bind to a library adapter comprised in library fragments.
- Embodiment 181 The method of any one of embodiments 177-179 or the solid support of embodiment 180, wherein at least one unwanted RNA sequence has at least 90%, at least 95%, or at least 99% homology to a high-abundance RNA sequence in a sample used to prepare the library of fragments.
- Embodiment 182 The method or solid support of embodiment 181, wherein the high-abundance RNA sequence is a ribosomal RNA (rRNA) sequence.
- rRNA ribosomal RNA
- Embodiment 183 The method or solid support of embodiment 182, wherein the unwanted RNA sequence is globin mRNA or 28S, 23S, 18S, 5.8S, 5S, 16S, 12S, HBA-A1, HBA-A2, HBB, HBB-B1, HBB-B2, HBG1, or HBG2 RNA, or a fragment thereof.
- Embodiment 184 The method or solid support of any one of embodiments 177-183, wherein each pool of immobilized oligonucleotides comprises 2 or more, 5 or more, 10 or more, 25 or more, 50 or more, 100 or more, 200 or more, 300 or more, 400 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, 1000 or more, or 1100 or more oligonucleotides.
- Embodiment 185 The solid support of any one of embodiments ISO- 184, wherein adapter complements that are all or partially complementary to the solid support adapter sequences are bound to the solid support adapter sequences of the second pool and wherein the binding of the adapter complements to the solid support adapter sequences is reversible.
- Embodiment 186 A method of amplifying desired cDNA library fragments from a library of cDNA fragments prepared from RNA, comprising (a) providing the solid support of embodiment 185; (b) adding the library of fragments to the solid support and hybridizing the library fragments to at least one immobilized oligonucleotide to allow binding of unwanted library fragments to the first pool of oligonucleotides; (c) collecting library fragments not bound to the first pool of oligonucleotides to prepare collected library fragments; (d) denaturing and removing library fragments bound to the first pool of oligonucleotides and adapter complements bound to the adapter sequences of the second pool of oligonucleotides; (e) adding the collected library fragments to the solid support and hybridizing the library fragments to at least one immobilized oligonucleotide to allow binding of desired library fragments to the second pool of oligonucleotides; and (f) amplifying
- Embodiment 187 A method for depleting unwanted RNA molecules comprised in a patient microbiome sample, wherein the patient microbiome sample comprises at least one target RNA or DNA sequence and at least one unwanted RNA molecule, comprising (a) sequencing a plurality of probe-development microbiome samples to determine at least one unwanted RNA molecule comprising a bacterial ribosomal RNA (rRNA) sequence from sequencing data; (b) preparing a probe set comprising at least one DNA probe complementary to the at least one unwanted RNA molecule; (c) contacting the patient microbiome sample with the probe set to prepare DNA: RNA hybrids; and (d) contacting the DNA:RNA hybrids with a ribonuclease that degrades the RNA from the DNA:RNA hybrids, thereby degrading the unwanted RNA molecules in the patient microbiome sample to form a degraded mixture.
- rRNA bacterial ribosomal RNA
- Embodiment 188 A method for depleting unwanted RNA molecules comprised in a patient microbiome sample, wherein the patient microbiome sample comprises at least one target RNA or DNA sequence and at least one unwanted RNA molecule, comprising (a) contacting the patient microbiome sample with a probe set comprising at least one sequence comprising at least one of SEQ ID NOs: 1-1131 to prepare DNA:RNA hybrids; and (b) contacting the DNA:RNA hybrids with a ribonuclease that degrades the RNA from the DNA:RNA hybrids, thereby degrading the unwanted RNA molecules in the patient microbiome sample to form a degraded mixture.
- Embodiment 189 The method of embodiment 187 or embodiment 188, further comprising (a) degrading any remaining DNA probes by contacting the degraded mixture with a DNA digesting enzyme, optionally wherein the DNA digesting enzyme is DNase I, to form a DNA degraded mixture; and (b) separating the degraded RNA from the degraded mixture or the DNA degraded mixture.
- Embodiment 190 A composition comprising a probe set comprising (a) at least one DNA probe comprising at least one sequence comprising at least one of SEQ ID NOs: 1-1131; and (b) a ribonuclease capable of degrading RNA in an DNA:RNA hybrid.
- kits comprising a probe set comprising (a) at least one DNA probe comprising at least one sequence comprising at least one of SEQ ID NOs: 1- 1131; and (b) a ribonuclease capable of degrading RNA in an DNA:RNA hybrid.
- Embodiment 192 The kit of embodiment 191, comprising (a) a probe set comprising at least one DNA probe comprising at least one of SEQ ID NOs: 1-1131; (b) a ribonuclease; (c) a DNase; and (d) RNA purification beads.
- Embodiment 193 The method of any one of embodiments 177-179, 181 or 186-189, the solid support of any one of embodiments 180-185, the composition of embodiment 190, or the kit of embodiment 191 or 192, wherein the pool of oligonucleotides or the probe set comprises 2 or more, 5 or more, 10 or more, 25 or more, 50 or more, 100 or more, 200 or more, 300 or more, 400 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, 1000 or more, 1100 or more, or 1131 sequences selected from SEQ ID NOs: 1-1131.
- Embodiment 194 The method of any one of embodiments 177-179, 181-184, or 186-189, the solid support of any one of embodiments 180-185, the composition of embodiment 190, or the kit of embodiment 191 or 192, wherein the pool of oligonucleotides or the probe set comprises at least one sequence comprising at least one of SEQ ID NOs: 1-10, 12-18, 21, 22, 24-33, 35, 39-43, 45-48, 50-73, 75, 77, 78, 81-84, 86-103, 105-107, 109-127, 129-134, 136-140, 142, 143, 148, 149, 151-155, 157, 160-165, 168-174, 176-182, 184-187, 189-194, 196-204, 206, 208-215, 217-223, 225, 227-246, 248-265, 269, 270, 272-277, 279, 281, 282, 284-290, 29
- Embodiment 195 The method, solid support, composition, or kit of embodiment 194, wherein the pool of oligonucleotides or the probe set comprises 100 or more, 500 or more, or 1000 or more sequences comprising at least one of SEQ ID NOs: 1-10, 12-18, 21, 22, 24-33, 35, 39-43, 45-48, 50-73, 75, 77, 78, 81-84, 86-103, 105-107, 109-127, 129-134, 136-140, 142, 143, 148, 149, 151-155, 157, 160-165, 168-174, 176-182, 184-187, 189-194, 196-204, 206, 208-215, 217-223, 225, 227-246, 248-265, 269, 270, 272-277, 279, 281, 282, 284-290, 292-301, 303-321, 323-331, 333-336, 338, 340-342, 344, 34
- Embodiment 196 The method, solid support, composition, or kit of embodiment 195, wherein the pool of oligonucleotides or the probe set comprises a sequence comprising each of SEQ ID NOs: 1-10, 12-18, 21, 22, 24-33, 35, 39-43, 45-48, 50-73, 75, 77, 78, 81-84, 86-103, 105-107, 109-127, 129-134, 136-140, 142, 143, 148, 149, 151-155, 157, 160-165, 168-174, 176-182, 184-187, 189-194, 196-204, 206, 208-215, 217-223, 225, 227- 246, 248-265, 269, 270, 272-277, 279, 281, 282, 284-290, 292-301, 303-321, 323-331, 333- 336, 338, 340-342, 344, 346-349, 351-355, 357
- Embodiment 197 The method, solid support, composition, or kit of any one of embodiments 194-196, wherein the pool of oligonucleotides or the probe set further comprises at least one sequence comprising at least one of SEQ ID NOs: 19, 74, 76, 85, 104, 108, 158, 175, 226, 278, 322, 339, 389, 513, 517, 520, 546, 553, 609, 611, 650, 662, 677, 683, 686, 706, 780, 827, 835, 882, 1022, 1059, 1077, 1078, 1098, 1106, 1111, 1114, 1128, and 1131.
- Embodiment 198 The method, solid support, composition, or kit of embodiment 197, wherein the pool of oligonucleotides or the probe set comprises 10 or more, 20 or more, or 30 or more sequences comprising at least one of SEQ ID NOs: 19, 74, 76, 85, 104, 108, 158, 175, 226, 278, 322, 339, 389, 513, 517, 520, 546, 553, 609, 611, 650, 662, 677, 683, 686, 706, 780, 827, 835, 882, 1022, 1059, 1077, 1078, 1098, 1106, 1111, 1114, 1128, and 1131.
- Embodiment 199 The method, solid support, composition, or kit of embodiment 198, wherein the pool of oligonucleotides or the probe set comprises a sequence comprising each of SEQ ID NOs: 19, 74, 76, 85, 104, 108, 158, 175, 226, 278, 322, 339, 389, 513, 517, 520, 546, 553, 609, 611, 650, 662, 677, 683, 686, 706, 780, 827, 835, 882, 1022, 1059, 1077, 1078, 1098, 1106, 1111, 1114, 1128, and 1131.
- Embodiment 200 The method of any one of embodiments 177-179, 181-184, or 186-189, the solid support of any one of embodiments 180-185, the composition of embodiment 190, or the kit of embodiment 191 or 192, wherein pool of oligonucleotides or the probe set comprises at least one sequence comprising at least one of SEQ ID NOs: 1- 10, 12-19, 21, 22, 24-33, 35, 39-43, 45-48, 50-78, 81-127, 129-134, 136-140, 142, 143, 148, 149, 151-155, 157, 158, 160-165, 168-182, 184-187, 189-194, 196-204, 206, 208-215, 217- 223, 225-246, 248-265, 269, 270, 272-279, 281, 282, 284-290, 292-301, 303-331, 333-336, 338-342, 344, 346-349, 351-355, 357
- Embodiment 201 The method, solid support, composition, or kit of embodiment 200, wherein the pool of oligonucleotides or the probe set comprises 100 or more, 500 or more, or 1000 or more sequences comprising at least one of SEQ ID NOs: 1-10, 12-19, 21, 22, 24-33, 35, 39-43, 45-48, 50-78, 81-127, 129-134, 136-140, 142, 143, 148, 149, 151-155, 157, 158, 160-165, 168-182, 184-187, 189-194, 196-204, 206, 208-215, 217-223, 225-246, 248-265, 269, 270, 272-279, 281, 282, 284-290, 292-301, 303-331, 333-336, 338- 342, 344, 346-349, 351-355, 357-359, 361-372, 374-380, 383-386, 388-391, 393, 395-401,
- Embodiment 202 The method, solid support, composition, or kit of embodiment 201, wherein the pool of oligonucleotides or the probe set comprises a sequence comprising each of SEQ ID NOs: 1-10, 12-19, 21, 22, 24-33, 35, 39-43, 45-48, 50-78, 81- 127, 129-134, 136-140, 142, 143, 148, 149, 151-155, 157, 158, 160-165, 168-182, 184-187, 189-194, 196-204, 206, 208-215, 217-223, 225-246, 248-265, 269, 270, 272-279, 281, 282, 284-290, 292-301, 303-331, 333-336, 338-342, 344, 346-349, 351-355, 357-359, 361-372, 374-380, 383-386, 388-391, 393, 395-401, 403-406, 408-420, 422-4
- Embodiment 203 The method, solid support, composition, or kit of any one of embodiments 194-202, wherein the pool of oligonucleotides or the probe set further comprises at least one sequence comprising at least one of SEQ ID NOs: 11, 20, 23, 34, 36-38, 44, 49, 79, 80, 128, 135, 141, 144-147, 150, 156, 159, 166, 167, 183, 188, 195, 205, 207, 216, 224, 247, 266-268, 271, 280, 283, 291, 302, 332, 337, 343, 345, 350, 356, 360, 373, 381, 382, 387, 392, 394, 402, 407, 421, 440, 442, 445, 461, 467, 470, 472, 478, 503,
- Embodiment 204 The method, solid support, composition, or kit of embodiment 203, wherein the pool of oligonucleotides or the probe set comprises 10 or more, 20 or more, or 30 or more sequences comprising at least one of SEQ ID NOs: 11, 20, 23, 34, 36-38, 44, 49, 79, 80, 128, 135, 141, 144-147, 150, 156, 159, 166, 167, 183, 188, 195, 205, 207, 216, 224, 247, 266-268, 271, 280, 283, 291, 302, 332, 337, 343, 345, 350, 356, 360, 373, 381, 382, 387, 392, 394, 402, 407, 421, 440, 442, 445, 461, 467, 470, 472, 478, 503, 515,
- Embodiment 205 The method, solid support, composition, or kit of embodiment 204, wherein the pool of oligonucleotides or the probe set comprises a sequence comprising each of SEQ ID NOs: 11, 20, 23, 34, 36-38, 44, 49, 79, 80, 128, 135, 141, 144- 147, 150, 156, 159, 166, 167, 183, 188, 195, 205, 207, 216, 224, 247, 266-268, 271, 280, 283, 291, 302, 332, 337, 343, 345, 350, 356, 360, 373, 381, 382, 387, 392, 394, 402, 407, 421,
- FIG. 1 provides an overview of a method of depleting unwanted library fragments derived from rRNA transcripts.
- a solid support such as a flowcell, comprises at least one immobilized oligonucleotide comprising a tether to attach the oligonucleotide to the solid support.
- the immobilized oligonucleotide could comprise the complement of a sequence that would be comprised in a library fragment comprising an insert of cDNA prepared from the rRNA (as labeled as “rRNA complement”).
- the library fragments can be flowed over the solid support, with fragment prepared from rRNA (i.e., library fragments comprising “rRNA library” sequence) hybridizing to immobilized oligonucleotides each comprising an rRNA complement.
- Library fragments that do not bind to the immobilized oligonucleotides can be siphoned for collection, and hybridized library fragments (i.e., unwanted library fragments) can then be denatured and siphoned to a waste container.
- the library fragments siphoned for collection can then be flowed over the solid support again to allow for binding of any additional unwanted library fragments, and steps of
- hybridizing unwanted library fragments, (2) collecting unbound library fragments, and (3) denaturing hybridized library fragment can be repeated, until a final set of collected unbound library fragments are collected that represent a library depleted of unwanted library fragments prepared from rRNA.
- Similar methods can be used for enrichment, wherein desired library fragments are bound to immobilized oligonucleotides comprising complementary sequences to these desired library fragments, except in the similar method the bound library fragments are used for sequencing and the library fragments that do not bind are siphoned for waste.
- Figure 2 shows an overview of a method for depleting unwanted library fragments and performing bridge amplification on the same solid support.
- the solid support used for this method would comprise immobilized oligonucleotides each comprising an rRNA complement, as well as immobilized oligonucleotides comprising adapter sequences that can bind to adapters comprised in library fragments.
- Such adapters comprised in immobilized oligonucleotides may be termed “solid support adapter sequences” and library fragments may comprise “library adapter sequences” that are all or partially complementary to the solid support adapter sequences.
- Solid support adapters may comprise as a P5 adapter sequence (SEQ ID NO: 1132) or a P7adapter sequence (SEQ ID NO: 1133), and/or their complements.
- Immobilized oligonucleotides comprising solid support adapter sequences may be bound to adapter complements that are all or partially complementary to the solid support adapter sequences, wherein the adapter complements hybridize to form double-stranded nucleic acid with the solid support adapter sequences.
- This hybridization inhibits binding of immobilized oligonucleotides comprising adapter sequences to library fragments (i.e., inhibits binding of solid support adapter sequences to library adapter sequences).
- the fragments prepared from rRNA can bind to immobilized oligonucleotides each comprising an rRNA complement, as described in the legend for Figure 1.
- desired library fragments unbound to immobilized oligonucleotides each comprising an rRNA complement
- unwanted library fragments and adapter complements can be denatured and siphoned to waste.
- the collected library fragments (comprising desired library fragments) can then be flowed over the flowcell and bound to the immobilized oligonucleotides comprising solid support adapter sequences by hybridization of library adapter sequences to solid support adapter sequences. Bridge amplification of bound library fragments can then be performed.
- the resulting amplified, depleted library could be sequenced, optionally after quantification and quality control.
- Figure 3 shows results on human gut microbiome rRNA depletion using the RiboZero method with RNase and standard probes (DPI) or human microbiome probes as described herein (HM, comprising HMvl and HMv2 probes). Significantly more rRNA depletion was seen with the HM probes.
- Figure 4 shows results on rRNA depletion from wastewater samples for RiboZero depletion with HM probes or “Mock” depletion that did not include probes. Significantly more rRNA depletion was seen with the HM probes.
- Bac bacterial rRNA
- Arc archaea rRNA
- Euk eukaryotic rRNA
- Rfam non-coding RNA as defined by Rfam database.
- Figure 5 shows results with a skin microbiome whole cell mix (ATCC MSA-2005TM).
- the experiment compared results with the RiboZero RNase protocol (either with standard DPI probes or with human microbiome HM probes) to those with the RiboZero-Bact that uses a probe-based hybridization approach to capture and deplete bacterial rRNAs from E. coli and B. subtilis.
- Table 1 provides a listing of certain sequences referenced herein.
- solid supports can be prepared for enriching desired library fragments or depleting unwanted library fragments, wherein at least oligonucleotide is immobilized to the solid support.
- the solid support is a flowcell.
- the at least one immobilized oligonucleotide comprises a sequence comprising any one or more of SEQ ID NOs: 1-1131.
- the at least one immobilized oligonucleotide comprises 2 or more, 5 or more, 10 or more, 25 or more, 50 or more, 100 or more, 200 or more, 300 or more, 400 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, 1000 or more, 1100 or more, or 1131 sequences selected from SEQ ID NOs: 1-1131 or its complement.
- the at least one immobilized oligonucleotide comprises 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, 1000 or more, 1100 or more, or 1131 sequences selected from SEQ ID NOs: 1-1131 or its complement.
- the at least one immobilized oligonucleotide comprises at least one sequence from a bacterial ribosomal RNA (rRNA) or its complement.
- the at least one immobilized oligonucleotide comprises at least one sequence comprising at least one of SEQ ID NOs: 1-10, 12-18, 21, 22, 24-33, 35, 39-43, 45- 48, 50-73, 75, 77, 78, 81-84, 86-103, 105-107, 109-127, 129-134, 136-140, 142, 143, 148, 149, 151-155, 157, 160-165, 168-174, 176-182, 184-187, 189-194, 196-204, 206, 208-215, 217-223, 225, 227-246, 248-265, 269, 270, 272-277, 279, 281, 282, 284-290, 292-301, 303- 321, 323-331
- the at least one immobilized oligonucleotide comprises 100 or more, 500 or more, or 1000 or more sequences comprising at least one of SEQ ID NOs: 1-10, 12-18, 21, 22, 24-33, 35, 39-43, 45-48, 50-73, 75, 77, 78, 81-84, 86-103, 105-107, 109-127, 129-134, 136-140, 142, 143, 148, 149, 151-155, 157, 160-165, 168-174, 176-182, 184-187, 189-194, 196-204, 206, 208-215, 217-223, 225, 227-246, 248-265, 269, 270, 272-277, 279, 281, 282, 284-290, 292-301, 303-321, 323-331, 333-336, 338, 340-342, 344, 346-349, 351-355, 357-359, 361-372, 3
- the at least one immobilized oligonucleotide comprises a sequence comprising each of SEQ ID NOs: 1-10, 12-18, 21, 22, 24-33, 35, 39-43, 45-48, 50-73, 75, 77, 78, 81-84, 86-103, 105- 107, 109-127, 129-134, 136-140, 142, 143, 148, 149, 151-155, 157, 160-165, 168-174, 176- 182, 184-187, 189-194, 196-204, 206, 208-215, 217-223, 225, 227-246, 248-265, 269, 270, 272-277, 279, 281, 282, 284-290, 292-301, 303-321, 323-331, 333-336, 338, 340-342, 344, 346-349, 351-355, 357
- the at least one immobilized oligonucleotide comprises at least one sequences of B. Bifidum rRNA or its complement. In some embodiments, the at least one immobilized oligonucleotide further comprises at least one sequence comprising at least one of SEQ ID NOs: 19, 74, 76, 85, 104, 108, 158, 175, 226, 278, 322, 339, 389, 513, 517, 520, 546, 553, 609, 611, 650, 662, 677, 683, 686, 706, 780, 827, 835, 882, 1022, 1059, 1077, 1078, 1098, 1106, 1111, 1114, 1128, and 1131 or its complement.
- the at least one immobilized oligonucleotide comprises 10 or more, 20 or more, or 30 or more sequences comprising at least one of SEQ ID NOs: 19, 74, 76, 85, 104, 108, 158, 175, 226, 278, 322, 339, 389, 513, 517, 520, 546, 553, 609, 611, 650, 662, 677, 683, 686, 706, 780, 827, 835, 882, 1022, 1059, 1077, 1078, 1098, 1106, 1111,
- the at least one immobilized oligonucleotide comprises a sequence comprising each of SEQ ID NOs: 19, 74, 76, 85, 104, 108, 158, 175, 226, 278, 322, 339, 389, 513, 517, 520, 546, 553, 609, 611, 650, 662, 677, 683, 686, 706, 780, 827, 835, 882, 1022, 1059, 1077, 1078, 1098, 1106, 1111, 1114, 1128, and 1131 or its complement.
- the at least one immobilized oligonucleotide comprises 100 or more, 500 or more, or 1000 or more sequences comprising at least one of SEQ ID NOs: or its complement. In some embodiments, the at least one immobilized oligonucleotide comprises a sequence comprising each of SEQ ID NOs: or its complement.
- the at least one immobilized oligonucleotide further comprises at least one sequence comprising at least one of SEQ ID NOs: 11, 20, 23, 34, 36-38, 44, 49, 79, 80, 128, 135, 141, 144-147, 150, 156, 159, 166, 167, 183, 188, 195, 205, 207, 216, 224, 247, 266-268, 271, 280, 283, 291, 302, 332, 337, 343, 345, 350, 356, 360, 373, 381, 382, 387, 392, 394, 402, 407, 421, 440, 442, 445, 461, 467, 470, 472, 478, 503,
- the at least one immobilized oligonucleotide comprises 10 or more, 20 or more, or 30 or more sequences comprising at least one of SEQ ID NOs: 11, 20, 23, 34, 36-38, 44, 49, 79, 80, 128, 135, 141, 144-147, 150, 156, 159, 166, 167, 183, 188, 195, 205, 207, 216, 224, 247, 266-268, 271, 280, 283, 291, 302, 332, 337, 343, 345, 350, 356, 360, 373, 381, 382, 387, 392, 394, 402, 407, 421, 440, 442,
- the at least one immobilized oligonucleotide comprises a sequence comprising each of SEQ ID NOs: 11, 20, 23, 34, 36-38, 44, 49, 79, 80, 128, 135, 141, 144-147, 150, 156, 159, 166, 167, 183, 188, 195, 205, 207, 216, 224, 247, 266-268, 271, 280, 283, 291, 302, 332,
- compositions comprising a library fragment bound to an immobilized oligonucleotide on a solid support.
- a singlestranded library fragment comprising cDNA prepared from a sample comprising RNA is hybridized to a solid support comprising immobilized oligonucleotides.
- the cDNA comprised in the composition is complementary to RNA comprised in the sample.
- kits for depleting or enriching libraries comprises a solid support disclosed herein and instructions for using the solid support.
- a kit may further comprise reagents for preparing a cDNA library from RNA, such as reagents for a stranded method of cDNA preparation from a sample comprising RNA, as described below.
- a wide variety of solid supports may be used to immobilize oligonucleotides for depleting or enriching as described herein, including those described in WO 2014/108810, which is incorporated in its entirety herein.
- the composition and geometry of the solid support can vary with its use.
- the solid support is a planar structure such as a slide, chip, microchip and/or array.
- the surface of a substrate can be in the form of a planar layer.
- the solid support comprises one or more surfaces of a flowcell.
- flowcell refers to a chamber comprising a solid surface across which one or more fluid reagents can be flowed.
- a flowcell is comprised within an apparatus or device for sequencing nucleic acids, which may be referred to as a sequencer.
- a sequence may also comprise reservoirs for collection of samples or tubing (such as for collecting samples in a reservoir of for exiting of waste).
- one or more reservoirs are separate from the flowcell and are comprised in the sequencer.
- modifications are made to standard sequencers to improve fluidics system recipes and/or hardware for use of reservoirs in the present methods.
- a “flowcell” may comprise a flowcell-like device that is not intended to be imaged. While standard flowcells used for imaging may be employed in the present methods, flowcells can also be engineered differently than flowcells intended for imaging.
- a flowcell may have a high density of immobilized oligonucleotides, wherein imaging infrastructure would have difficulty separating out into different bridge-amplified clusters associated with different immobilized oligonucleotides.
- a high density of immobilized oligonucleotides improves hybridization efficiency.
- standard clear glass may be used in a flowcell.
- hard plastic may be used in the flowcell.
- immobilized oligonucleotides are embedded in a substrate other than that of a standard flowcell (i. e. , embedded in a substrate other than PAZAM) to improve immobilization of oligonucleotides of longer length.
- unwanted RNA or “an unwanted RNA sequence” refers to any RNA that a user does not wish to analyze.
- an unwanted RNA includes the complement of an unwanted RNA sequence.
- RNA is converted into cDNA and this cDNA is prepared into a library, a user would sequence library fragments that were prepared from all RNA transcripts in the absence of enrichment or depletion. Methods described herein for depleting library fragments prepared from unwanted RNA can thus save the user time and consumables related to sequencing and analyzing sequencing data prepared from unwanted RNA.
- unwanted RNA or “unwanted RNA sequence” also includes fragments of such RNA.
- an unwanted RNA may comprise part of the sequence of an unwanted RNA.
- unwanted RNA sequence is from human, rat, mouse, or bacteria.
- the bacteria are Archaea species, E. Coli, or B. subtilis.
- unwanted library fragments refers to library fragments prepared from cDNA prepared from unwanted RNA.
- the unwanted RNA sequence comprises any one or more of SEQ ID NOs: 1-1131.
- unwanted RNA sequences are immobilized to a solid support.
- a range of different types of RNA may be unwanted.
- the unwanted RNA is high-abundance RNA.
- High-abundance RNA is RNA that is very abundant in many samples and which users do not wish to sequence, but it may or may not be present in a given sample.
- the high-abundance RNA sequence is a ribosomal RNA (rRNA) sequence.
- rRNA ribosomal RNA
- Exemplary high- abundance RNA are disclosed in WO2021/127191 and WO 2020/132304, each of which is incorporated by reference herein in its entirety.
- the high-abundance RNA sequences are the most abundant RNA sequences determined to be in a sample. In some embodiments, the high-abundance RNA sequences are the most abundant RNA sequences across a plurality of samples even though they may not be the most abundant in a given sample. In some embodiments, a user utilizes a method of determining the most abundant RNA sequences in a sample, as described herein.
- the most abundant sequences are the 100 most abundant sequences.
- the method also is capable of depleting the 1,000 most abundant sequences, or the 10,000 most abundant sequences in a sample.
- the unwanted RNA sequence comprises a sequence with homology of at least 90%, at least 95%, or at least 99% to a most abundant sequence in a sample comprising RNA.
- the unwanted RNA sequence comprises a sequence with homology of at least 90%, at least 95%, or at least 99% to a most abundant sequence in a sample comprising RNA, wherein the most abundant sequences comprise the 100 most abundant sequences.
- homology is measured against the 1,000 most abundant sequences, or the 10,000 most abundant sequences.
- the high-abundance RNA sequences are comprised in RNA known to be highly abundant in a range of samples.
- the unwanted RNA sequence is globin mRNA or 28S, 23S, 18S, 5.8S, 5S, 16S, 12S, HBA-A1, HBA-A2, HBB, HBB-B1, HBB-B2, HBG1, or HBG2 RNA, or a fragment thereof.
- the unwanted RNA sequence is 28S, 18S, 5.8S, 5S, 16S, or 12S RNA from humans, or a fragment thereof.
- the unwanted RNA sequence is rat 16S, rat 28S, mouse 16S, or mouse 28S RNA.
- the unwanted RNA sequence is comprised in mRNA related to one or more “housekeeping” genes.
- a housekeeping gene may be one that is commonly expressed in a sample from a tumor or other oncology-related sample, but that is not implicated in tumor genesis or progression
- the unwanted RNA sequence is comprised in 23 S, 16S, or 5S RNA from Gram-positive or Gram-negative bacteria. In some embodiments, the unwanted RNA sequence is from an organism in the human microbiome.
- the unwanted RNA sequence is comprised in a host transcriptome.
- a user may wish to study library fragments prepared from RNA from organisms comprised in the human microbiome, without analyzing library fragments prepared from human RNA.
- RNA or “a desired RNA sequence” refers to any RNA that a user wants to analyze.
- a desired RNA includes the complement of a desired RNA sequence.
- Desired RNA may be RNA from which a user would like to collect sequencing data, after cDNA and library preparation.
- the desired RNA is mRNA (or messenger RNA).
- the desired RNA is a portion of the mRNA in a sample.
- a user may want to analyze RNA transcribed from cancer-related genes, and thus this is the desired RNA.
- a user may wish to analyze RNA from organisms comprised in a human microbiome, and thus RNA from organisms comprised in the human microbiome is the desired RNA and human RNA is the unwanted RNA.
- verified library fragments refers to library fragments prepared from cDNA prepared from desired RNA.
- the desired RNA sequence is an exome sequence. In some embodiments, the present methods are for exome enrichment. [00266] In some embodiments, the desired RNA sequence is from human, rat, mouse, and/or bacteria. In some embodiments, the desired RNA sequence is from an organism in the human microbiome.
- oligonucleotides for enriching or depleting are immobilized to a solid support. Such immobilized oligonucleotides may be referred to as tethered to the solid support. In some embodiments, the oligonucleotide may be immobilized to the solid support via a linker molecule.
- immobilized and attached are used interchangeably herein and both terms are intended to encompass direct or indirect, covalent or non-covalent attachment, unless indicated otherwise, either explicitly or by context.
- covalent attachment may be preferred, but generally all that is required is that the at least one immobilized oligonucleotide remains immobilized or attached to the support under the conditions in which it is intended to use the support, for example for enriching or depleting.
- a “tether” refers to any means of immobilizing an oligonucleotide to a solid support.
- a solid support such as a flowcell
- a flowcell contains a polymer coating.
- the covalently attached polymer is PAZAM.
- the polymer coating comprises reactive sites for reacting with oligonucleotides, such as oligonucleotides described herein.
- covalently attached polymers are described in WO 2013/184796, which is incorporated by reference in its entirety herein.
- a polymer such as PAZAM is crosslinked using ultraviolet light.
- immobilized oligonucleotides may be designed to comprise a cleavage site.
- a method may comprise a step to cleave immobilized oligonucleotides to remove them from the solid support.
- the resulting fragments from the immobilized oligonucleotides are collected in a waste container comprised in a sequencer.
- a tether may comprise a cleavage site.
- immobilized oligonucleotides described herein are single-stranded. In this way, the immobilized oligonucleotides are available to hybridize to single-stranded library fragments that are all of partially complementary to a sequence comprised in the immobilized oligonucleotides.
- immobilized oligonucleotides One skilled in the art could design the length of immobilized oligonucleotides to allow for their preferred level of affinity for the interaction between immobilized oligonucleotides and library fragments that are all or partially complementary (i.e., longer immobilized oligonucleotides would be expected to exhibit higher affinity binding to single-stranded library fragments that are all or partially complementary).
- a sequence comprised in an immobilized oligonucleotide can be partly or completely complementary to the sequence of a library fragment prepared from an unwanted RNA for depletion. In some embodiments, a sequence comprised in an immobilized oligonucleotide can be partly or completely complementary to the sequence of a library fragment prepared from a desired RNA for enrichment.
- each immobilized oligonucleotide is from 10 to 100 nucleotides long, from 20 to 100 nucleotides long, from 20 to 80 nucleotides long, from 40 to 60 nucleotides long, from 45 to 55 nucleotides long, or 50 nucleotides long.
- the at least one immobilized oligonucleotide is 45-55 bases in length, optionally wherein the at least one immobilized oligonucleotide is 50 bases in length.
- an immobilized oligonucleotide has a molecular weight (M.W.) of 15,000 to 15,500 Daltons.
- multiple different oligonucleotides comprising a sequence all or partially complementary to an unwanted or desired RNA may be immobilized on a solid support.
- these multiple different oligonucleotides are all or partially complementary to different sequences comprised in an unwanted or desired RNA. For example, if a user wants to deplete a given rRNA, the user may prepare multiple oligonucleotides with overlapping or non-overlapping sequences corresponding to this rRNA.
- having multiple immobilized oligonucleotides corresponding to different sequences from a given unwanted RNA can improve efficiency of depleting of library fragments prepared from this RNA.
- having multiple immobilized oligonucleotides corresponding to different sequences from a given desired RNA can improve efficiency of enrichment of library fragments prepared from this RNA.
- this improved efficiency may be because library fragments may be generated randomly from cDNA prepared from a given RNA, and a user cannot predict the specific insert sequence of cDNA comprised in a given fragment.
- a sequence comprised in an immobilized oligonucleotide can be completely or partially complementary to a particular location on the RNA to be depleted or enriched (i. e. , a target location), for example the sequence comprised in an immobilized oligonucleotide can be at least 80%, 85%, 90%, 95%, or 100% complementary, or any range in between, to a target location on an RNA transcript to be depleted or enriched.
- immobilized oligonucleotides may bind to a set of different sequences comprised in an RNA to be depleted.
- multiple immobilized oligonucleotides may be designed that tile an RNA sequence intended for depletion, such as the tiling described in WO 2020132304, which is incorporated herein in its entirety.
- multiple immobilized oligonucleotides designed against a target sequence can increase the likelihood of binding of a fragment generated from the target sequence to at least one immobilized oligonucleotide.
- library inserts comprised in library fragments may comprise approximately 150bp, and the immobilized oligonucleotides described herein may comprise 50-80 nucleotides.
- an immobilized oligonucleotide designed to bind an adjacent target sequence may likely be able to hybridize to the fragment. In this way, tiling of sequences can increase the likelihood of successful depletion or enrichment of fragments prepared from an RNA sequence.
- the present oligonucleotides comprise modified or unmodified nucleic acid.
- a “modified nucleic acid” refers to any substitution from a naturally occurring nucleic acid.
- a modified nucleic acid may comprise one or more modifications to the sugar-phosphate backbone or the pendant base groups. Such modifications can improve stability of immobilized oligonucleotides.
- one, at least one, or each of the one or more immobilized nucleic acids comprises RNA, deoxyribonucleic acid (DNA), xeno nucleic acid (XNA), or a combination thereof.
- the XNA can comprise 1,5-anhydrohexitol nucleic acid (HNA), cyclohexene nucleic acid (CeNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), Fluoro Arabino nucleic acid (FANA), or a combination thereof.
- an immobilized nucleic acid consists of modified nucleic acids.
- a certain percentage of the nucleic acids comprised in an immobilized nucleic acid are modified nucleic acids, for example every third nucleotide may be a modified nucleic acid.
- the at least one immobilized oligonucleotide comprises the sequence or complementary sequence of an unwanted RNA.
- Solid supports with such immobilized oligonucleotides comprising the sequence or complementary sequence of unwanted RNA may be used for depleting library fragments prepared from unwanted RNA using methods described herein.
- the at least one immobilized oligonucleotide comprises the sequence or complementary sequence of a desired RNA.
- Solid supports with such immobilized oligonucleotides comprising the sequence or complementary sequence of desired RNA may be used for enriching library fragments prepared from desired RNA using methods described herein.
- oligonucleotides for depleting comprise one or more unwanted RNA sequence.
- immobilized oligonucleotides are designed to deplete unwanted library fragments from a library.
- the unwanted library fragments comprise library fragments prepared from unwanted RNA.
- a representative example of a solid support with immobilized oligonucleotides for depleting unwanted library fragments is shown in Figure 1.
- immobilized oligonucleotides are designed to deplete each of most abundant species that are determined from a sample.
- RNA RiboZero+ probes and nuclease-based depletion of abundant transcripts using the RiboZero+ probes have been described in WO 2020/132304A1, the content of which is incorporated by reference in its entirety.
- immobilized oligonucleotides are designed for depleting abundant transcripts described in WO 2020/132304A1.
- unwanted RNA sequences are determined by assessing sequencing results to determine abundant sequences in a sample comprising RNA.
- the unwanted RNA sequences are selected by determining the most abundant sequences in a sample comprising RNA.
- the most abundant sequences comprise the 100 most abundant sequences, the 1,000 most abundant sequences, or the 10,000 most abundant sequences.
- the unwanted RNA sequence comprises a sequence with homology of at least 90%, at least 95%, or at least 99% to a most abundant sequence in a sample comprising RNA.
- the unwanted RNA sequence comprises a sequence with homology of at least 90%, at least 95%, or at least 99% to a most abundant sequence in a sample comprising RNA, wherein the most abundant sequences comprise the 100 most abundant sequences, the 1,000 most abundant sequences, or the 10,000 most abundant sequences.
- WO 2021/127191 which is incorporated herein in its entirety, describes methods of selecting abundant regions from a sample comprising RNA.
- Immobilized oligonucleotides can be designed using methods from WO 2021/127191 of identifying abundant regions using standard publicly available software. In some embodiments, methods of identifying abundant regions can avoid bias towards known samples within an environmental sample.
- an exemplary type of immobilized oligonucleotides for use in depleting library fragments prepared from unwanted RNA is shown in Figure 1.
- the unwanted library fragments are prepared from rRNA and may be termed an “rRNA library.”
- the rRNA library comprises library fragments prepared from a first strand of cDNA prepared from RNA.
- an immobilized oligonucleotides may be an “rRNA complement” that can bind to the rRNA library.
- Immobilized oligonucleotides comprising an rRNA complement are one representative type of immobilized oligonucleotide for use in depleting, and one skilled in the art could design such oligonucleotides for depleting library fragments prepared from any type of unwanted RNA.
- unwanted RNA may be comprised in some immobilized oligonucleotides, and the complement of unwanted RNA may be comprised in other immobilized oligonucleotides.
- Table 1 describes a set of sequences that may comprised in immobilized oligonucleotides. Immobilized oligonucleotides or their complements listed in Table 2 may have particular use in studies of microbiome samples.
- the immobilized oligonucleotides listed in Table 2 were designed by sequencing total RNA derived from human fecal matter to identify abundant rRNA sequences that were detected using the publicly available rRNA classifier SortMeRNA (as described in Kopylova et al., Bioinformatics 28(24):3211-3217 (2012)). The most abundant transcripts were identified, and DNA probes were designed against these transcripts. The depletion has been tested with fecal, skin, oral and vaginal samples using the Total RNA stranded kit as well as with samples derived from various soil types with much better results in comparison to a standard depletion probe panel (data not shown).
- the oligonucleotides listed in Table 2 are designed to remove rRNA sequences from metatranscriptomics samples, such as stool, and are antisense to the rRNA sequence that they target.
- the at least one immobilized nucleotide comprises 2 or more, 5 or more, 10 or more, 25 or more, 50 or more, 100 or more, 200 or more, 300 or more, 400 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, 1000 or more, 1100 or more, or 1131 sequences selected from SEQ ID NOs: 1-1131 or its complement.
- the at least one immobilized nucleotide comprises 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, 1000 or more, 1100 or more, or 1131 sequences selected from SEQ ID NOs: 1-1131 or its complement.
- the at least one immobilized oligonucleotide comprises at least one sequence comprised in the HMvl sequences and comprising SEQ ID NOs: 1-10, 12-18, 21, 22, 24-33, 35, 39-43, 45-48, 50-73, 75, 77, 78, 81-84, 86-103, 105- 107, 109-127, 129-134, 136-140, 142, 143, 148, 149, 151-155, 157, 160-165, 168-174, 176- 182, 184-187, 189-194, 196-204, 206, 208-215, 217-223, 225, 227-246, 248-265, 269, 270, 272-277, 279, 281, 282, 284-290, 292-301, 303-321, 323-331, 333-336, 338, 340-342, 344, 346-349, 351-355, 357-359, 361-372, 374
- the at least one immobilized oligonucleotide comprises 100 or more, 500 or more, or 1000 or more sequences comprising at least one of SEQ ID NOs: 1-10, 12-18, 21, 22, 24-33, 35, 39-43, 45-48, 50-73, 75, 77, 78, 81-84, 86-103, 105-107, 109-127, 129-134, 136-140, 142, 143, 148, 149, 151-155, 157, 160-165, 168-174, 176-182, 184-187, 189-194, 196-204, 206, 208-215, 217-223, 225, 227-246, 248-265, 269, 270, 272-277, 279, 281, 282, 284-290, 292-301, 303-321, 323-331, 333-336, 338, 340-342, 344, 346-349, 351-355, 357-359, 361-372, 3
- the at least one immobilized oligonucleotide comprises a sequence comprising each of SEQ ID NOs: 1-10, 12-18, 21, 22, 24-33, 35, 39- 43, 45-48, 50-73, 75, 77, 78, 81-84, 86-103, 105-107, 109-127, 129-134, 136-140, 142, 143, 148, 149, 151-155, 157, 160-165, 168-174, 176-182, 184-187, 189-194, 196-204, 206, 208- 215, 217-223, 225, 227-246, 248-265, 269, 270, 272-277, 279, 281, 282, 284-290, 292-301, 303-321, 323-331, 333-336, 338, 340-342, 344, 346-349, 351-355, 357-359, 361-372, 374- 380, 383-386, 388
- the at least one immobilized oligonucleotide further comprises at least one sequence comprised in the HMv2 sequences and comprising at least one of SEQ ID NOs: 19, 74, 76, 85, 104, 108, 158, 175, 226, 278, 322, 339, 389, 513, 517, 520, 546, 553, 609, 611, 650, 662, 677, 683, 686, 706, 780, 827, 835, 882, 1022, 1059, 1077, 1078, 1098, 1106, 1111, 1114, 1128, and 1131 or its complement.
- the at least one immobilized oligonucleotide comprises 10 or more, 20 or more, or 30 or more sequences comprising at least one of SEQ ID NOs: 19, 74, 76, 85, 104, 108, 158, 175, 226, 278, 322, 339, 389, 513, 517, 520, 546, 553, 609, 611, 650, 662, 677, 683, 686, 706, 780, 827, 835, 882, 1022, 1059, 1077, 1078, 1098, 1106, 1111, 1114, 1128, and 1131 or its complement.
- the at least one immobilized oligonucleotide comprises a sequence comprising each of SEQ ID NOs: 19, 74, 76, 85, 104, 108, 158, 175, 226, 278, 322, 339, 389, 513, 517, 520, 546, 553, 609, 611, 650, 662, 677, 683, 686, 706, 780, 827, 835, 882, 1022, 1059, 1077, 1078, 1098, 1106, 1111, 1114, 1128, and 1131 or its complement.
- the at least one immobilized oligonucleotide further comprises at least one sequence comprised in the HM sequences (comprising both HMvl and HMv2 probes) and comprising at least one of SEQ ID NOs: 1-10, 12-19, 21, 22, 24-33, 35, 39-43, 45-48, 50-78, 81-127, 129-134, 136-140, 142, 143, 148, 149, 151-155, 157, 158, 160-165, 168-182, 184-187, 189-194, 196-204, 206, 208-215, 217-223, 225-246, 248- 265, 269, 270, 272-279, 281, 282, 284-290, 292-301, 303-331, 333-336, 338-342, 344, 346- 349, 351-355, 357-359, 361-372, 374-380, 383-386, 388-391, 393, 395-
- the at least one immobilized oligonucleotide comprises 10 or more, 20 or more, or 30 or more sequences comprising at least one of SEQ ID NOs: 1-10, 12-19, 21, 22, 24-33, 35, 39-43, 45-48, 50-78, 81-127, 129-134, 136-140, 142, 143, 148, 149, 151-155, 157, 158, 160-165, 168-182, 184-187, 189-194, 196-204, 206, 208- 215, 217-223, 225-246, 248-265, 269, 270, 272-279, 281, 282, 284-290, 292-301, 303-331, 333-336, 338-342, 344, 346-349, 351-355, 357-359, 361-372, 374-380, 383-386, 388-391, 393, 395-401, 403-406, 408-420, 422-439, 441, 443,
- the at least one immobilized oligonucleotide comprises a sequence comprising each of SEQ ID NOs: 1-10, 12-19, 21, 22, 24-33, 35, 39- 43, 45-48, 50-78, 81-127, 129-134, 136-140, 142, 143, 148, 149, 151-155, 157, 158, 160-165, 168-182, 184-187, 189-194, 196-204, 206, 208-215, 217-223, 225-246, 248-265, 269, 270, 272-279, 281, 282, 284-290, 292-301, 303-331, 333-336, 338-342, 344, 346-349, 351-355, 357-359, 361-372, 374-380, 383-386, 388-391, 393, 395-401, 403-406, 408-420, 422-439, 441, 443, 444, 446-460, 462-466, 468, 4
- the at least one immobilized oligonucleotide further comprises at least one sequence comprised in the DPI sequences and comprising at least one of SEQ ID NOs: 11, 20, 23, 34, 36-38, 44, 49, 79, 80, 128, 135, 141, 144-147, 150, 156, 159, 166, 167, 183, 188, 195, 205, 207, 216, 224, 247, 266-268, 271, 280, 283, 291, 302, 332, 337, 343, 345, 350, 356, 360, 373, 381, 382, 387, 392, 394, 402, 407, 421, 440, 442,
- the at least one immobilized oligonucleotide comprises 10 or more, 20 or more, or 30 or more sequences comprising at least one of SEQ ID NOs: 11, 20, 23, 34, 36-38, 44, 49, 79, 80, 128, 135, 141, 144-147, 150, 156, 159, 166, 167, 183, 188, 195, 205, 207, 216, 224, 247, 266-268, 271, 280, 283, 291, 302, 332, 337, 343, 345, 350, 356, 360, 373, 381, 382, 387, 392, 394, 402, 407, 421, 440, 442, 445, 461, 467,
- the at least one immobilized oligonucleotide comprises a sequence comprising each of SEQ ID NOs: 11, 20, 23, 34, 36-38, 44, 49, 79, 80, 128, 135, 141, 144-147, 150, 156, 159, 166, 167, 183, 188, 195, 205, 207, 216, 224, 247, 266-268, 271, 280, 283, 291, 302, 332, 337, 343, 345, 350, 356, 360, 373, 381, 382, 387, 392, 394, 402, 407, 421, 440, 442, 445, 461, 467, 470, 472, 478, 503, 515, 525, 530, 533, 534,
- immobilized oligonucleotides are designed for enriching desired library fragments.
- oligonucleotides for enriching comprise one or more desired RNA sequence.
- a user can design oligonucleotides for enriching using similar means of selecting probes as described above for depleting. For example, a user could prepare immobilized oligonucleotides of desired RNA sequences comprised in organisms of interest in the human microbiome, for use in enriching library fragments prepared from these desired RNA sequences. Likewise, a user could prepare immobilized oligonucleotides of desired mRNA sequences from an organism of interest.
- desired RNA may be comprised in some immobilized oligonucleotides, and the complement of desired RNA may be comprised in other immobilized oligonucleotides.
- solid supports comprise immobilized oligonucleotides comprising adapter sequences.
- the adapter sequences comprised in immobilized oligonucleotides are solid support adapter sequences.
- solid support adapter sequences refer to adapter sequences that are comprised in oligonucleotides immobilized to the solid support.
- solid support adapter sequences bind to library adapter sequences.
- library adapter sequence refers to an adapter sequence incorporated into library fragments, wherein the library adapter sequence can bind to the solid support adapter sequence.
- solid support adapter sequences can serve to immobilize library fragments to a solid support, wherein this immobilizing is not due to the cDNA sequence comprised in the library fragment, but due to binding to a library adapter comprised in library fragments.
- binding of a library adapter sequence comprised in a library fragment to a solid support adapter sequence comprised in an immobilized oligonucleotide serves to immobilize the library fragment to the solid support.
- library adapter sequences are incorporated into library fragments during library preparation.
- the library of fragments added to the solid support is prepared by a method comprising incorporating one or more library adapters that specifically bind to solid support adapter sequences comprising P5 (SEQ ID NO: 1132), P7 (SEQ ID NO: 1133), and/or their complements.
- Such methods for incorporating one or more library adapters may be tagmentation or fragmentation followed by adapter ligation.
- library adapter sequences are incorporated into library fragments after performing enriching or depleting as described herein. In other words, enriching or depleting may be performed, and then library adapters may be added to the enriched or depleted library. In some embodiments, library adapter sequences are added to collected library fragments. In some embodiments, the library adapter sequences are added to collected library fragments by ligation.
- library fragments comprise library adapters and the solid support comprises immobilized oligonucleotides comprising solid support adapter sequences that can bind to library adapters.
- the solid support adapter sequences comprise a P5 sequence (SEQ ID NO: 1132), a P7 sequence (SEQ ID NO: 1133), and/or their complements.
- library adapter sequences comprise a sequence complementary to P5 sequence or P7 sequence.
- library adapter sequences comprise a P5 sequence or P7 sequence.
- a solid support comprises immobilized oligonucleotides comprising P5 and/or its complement. In some embodiments, a solid support comprises immobilized oligonucleotides comprising P7 and/or its complement. In some embodiments, a solid support comprises more than one pool of immobilized oligonucleotides, wherein one or more pool may comprise immobilized oligonucleotides comprising a P5 sequence, a P7 sequence, and/or their complements.
- library adapter sequences comprised in library fragments specifically bind to solid support adapter sequences comprising P5 (SEQ ID NO: 1132), P7 (SEQ ID NO: 1133), and/or their complements.
- adapter complements can bind to solid support adapter sequences.
- an “adapter complement” is an oligonucleotide that can bind to a solid support adapter sequence.
- the solid support adapter sequence is single-stranded and the adapter complement is single-stranded.
- adapter complements that are all or partially complementary to the solid support adapter sequences are bound to the solid support adapter sequences.
- the binding of adapter complements to solid support adapter sequences serves to prevent binding of library adapter sequences comprised in library fragments to solid support adapter sequences.
- a user can control when library fragments can bind to solid support adapter sequences comprised in immobilized oligonucleotides.
- a user can block binding of library adapter sequences (using adapter complements) to solid support adapter sequences during enriching or depleting steps.
- adapter complements bound to the solid support adapter sequences generate double-stranded immobilized oligonucleotides.
- solid support adapter sequences bound to adapter complements cannot bind to library adapters.
- double-stranded immobilized oligonucleotides comprising a solid support adapter sequence and an adapter complement cannot bind to library adapter sequences.
- the binding of the adapter complements to the solid support adapter sequences is reversible.
- an increase in temperature or a denaturing agent can be used to denature library adapter sequences from the solid support adapter sequences.
- solid support adapter sequences comprised in immobilized oligonucleotides can be available for binding to library adapter sequences.
- a solid support comprises more than one pool of immobilized oligonucleotides on its surface.
- a solid support may comprise a first pool of immobilized oligonucleotides for depleting and a second pool of immobilized oligonucleotides for enriching.
- one pool of immobilized oligonucleotides may be blocked (such as with complementary nucleic acid sequences) to avoid binding to complementary library fragments during certain steps of methods using the solid support.
- blocking may be used to inhibit binding of P5/P7 sequences until a user wishes to perform bridge amplification after depletion/enrichment (as shown in Figure 2).
- a solid support has two pools of immobilized oligonucleotides on its surface, wherein the first pool comprises immobilized oligonucleotides each comprising an unwanted RNA sequence and the second pool comprises immobilized oligonucleotides each comprising a solid support adapter sequence that can bind to a library adapter comprised in library fragments (as shown in Figure 2).
- solid support adapter sequences are bound by adapter complements, wherein the adapter complements can be denatured during a method to allow binding of solid support adapter sequences to library adapters in library fragments.
- Such a solid support can be used for methods of preparing a depleted library and amplifying the depleted library on the same solid support (such as described in Example 2).
- At least one unwanted RNA sequence has at least 90%, at least 95%, or at least 99% homology to a high-abundance RNA sequence in a sample used to prepare the library of fragments. In some embodiments, all unwanted sequences have at least 90%, at least 95%, or at least 99% homology to a high-abundance RNA sequence in a sample used to prepare the library of fragments.
- a method selects cDNA library fragments from a library of cDNA fragments prepared from RNA. This selecting may be depleting unwanted library fragments by removing them, or this selecting may be enriching desired library fragments and collecting them. In some embodiments, selecting includes both depleting unwanted library fragments and enriching desired library fragments.
- a method of selecting cDNA library fragments from a library of cDNA fragments prepared from RNA comprises (a) preparing a solid support comprising a pool of immobilized oligonucleotides, wherein each immobilized oligonucleotide in the pool comprises a nucleic acid sequence corresponding to an RNA sequence or its complement, (b) adding the library of fragments to the solid support and hybridizing the library fragments to at least one immobilized oligonucleotide to allow binding of library fragments to at least one immobilized oligonucleotide, and (c) collecting library fragments either bound or not bound to at least one immobilized oligonucleotide.
- the selecting is depleting unwanted cDNA library fragments, wherein the RNA sequence comprises an unwanted RNA sequence, the unwanted library fragments comprise those prepared from unwanted RNA sequences, and the collecting comprises collecting library fragment not bound to at least one immobilized oligonucleotide.
- the selecting is enriching desired cDNA library fragments, wherein the RNA sequence comprises a desired RNA sequence, the desired library fragments comprise those prepared from desired RNA sequences, and the collecting comprises collecting library fragment bound to at least one immobilized oligonucleotide.
- the library of fragments is subjected to depleting unwanted cDNA library fragments and the collected library fragments not bound to at least one immobilized oligonucleotides are then subjected to enriching desired cDNA library fragments.
- the library fragments are prepared from a sample comprising RNA.
- library fragments are prepared from cDNA prepared from RNA in a sample.
- Such a sample may be any type comprising RNA and any method of cDNA and library preparation may be combined with the present method.
- the present methods using solid supports decrease library preparation costs and hands-on-time, as compared to prior art methods of depleting unwanted RNA, followed by library preparation.
- the present methods reduce degradation and/or loss of rare RNA transcripts that may be seen with RNase-H-mediated depletion methods that are performed before library preparation. Methods described herein can be used for depletion of unwanted rRNA transcripts, as well as unwanted non-rRNA transcripts (such as for depleting host transcriptome when evaluating microbiome samples).
- methods of depleting or enriching library fragments as described herein improves yield of the resulting library after the enriching or depleting in comparison to methods wherein RNA is depleted or enriched prior to library preparation. Such an improvement in yield may be due to the fact that library preparation itself can be limited when a starting RNA sample has very low concentrations of RNA.
- the present methods of enriching or depleting after library preparation can avoid or reduce the impact of low RNA concentration in the starting sample on library yield.
- the present methods of depleting and enriching are flexible for use with any upstream methods of cDNA and library preparation that a user prefers.
- a user can choose the best method of cDNA preparation and the best method of library preparation for their particular sample, and then the user can deplete or enrich the resulting library fragments using methods described herein.
- cDNA is prepared using a stranded method.
- library preparation comprises incorporating one or more adapter sequence into library fragments.
- one or more adapter sequence may be incorporated into fragments after the present methods of enriching or depleting.
- single-stranded library fragments are preparing before adding a library of fragments to a solid support. In this way, single-stranded library fragments can bind to single-stranded immobilized oligonucleotides on the surface of a solid support.
- the method is performed after library preparation from cDNA prepared from RNA. In some embodiments, the method does not require degradation of RNA.
- the library depleted of unwanted library fragments or enriched for desired library fragments is assessed for library size and/or concentration.
- the library depleted of unwanted library fragments or enriched for desired library fragments may also be amplified and/or sequenced.
- a method comprises steps of both depleting unwanted library fragments and enriching desired library fragments.
- a depletion flowcell may be used to deplete unwanted library fragments, and the depleted library can then be enriched for desired library fragments using an enrichment flowcell.
- Such a workflow comprising both depletion and enrichment may have particular use for generating data from desired library fragments that are relatively rare in a sample. For example, data from library fragments generated from a particular microorganism comprised in a metatranscriptomics sample may be improved by a method of depletion followed by enrichment.
- a method comprises amplifying and/or sequencing on the same flowcell used for depleting and/or enriching.
- Such a method comprising amplifying and/or sequencing on the same flowcell used for depleting and/or enriching may be termed a “one-pot” or “single flowcell” method.
- amplifying and sequencing are not performed on the flowcell used for depleting and/or enriching.
- a collected library may be amplified in a thermocycler and then the amplified library fragments are sequenced on a flowcell that is different from the flowcell used for the depleting and/or enriching.
- amplifying is performed on the flowcell used for depleting and/or enriching, and the amplified library fragments are then sequenced on a flowcell that is different from the flowcell used for the depleting and/or enriching.
- a method may comprise bridge amplification on the flowcell used for depleting and/or enriching (as described below), and amplified library fragments are then sequenced on a flowcell that is different from the flowcell used for the depleting and/or enriching.
- library fragments prepared from one or more abundant RNA transcripts, sequences thereof, or subsequences thereof have been depleted from the sample using a plurality of immobilized oligonucleotides after RNA transcripts are reverse transcribed to generate complementary DNAs (cDNAs) and library fragments are prepared from the cDNA.
- the library fragments are sequenced after the depleting to generate a plurality of sequence reads.
- the one or more abundant RNA transcripts can be ribosomal RNA transcripts and/or globin mRNA transcripts.
- a method of depleting unwanted cDNA library fragments from a library of cDNA fragments prepared from RNA comprises (a) preparing a solid support comprising at least one immobilized oligonucleotide, wherein each immobilized oligonucleotide comprises a nucleic acid sequence corresponding to an unwanted RNA sequence or its complement, (b) adding the library of fragments to the solid support and hybridizing the library fragments to the at least one immobilized oligonucleotide to allow binding of unwanted library fragments to the at least one immobilized oligonucleotide, and (c) collecting library fragments not bound to the at least one immobilized oligonucleotide.
- the solid support for depleting comprises a pool of oligonucleotides.
- the pool of oligonucleotides comprises 2 or more, 5 or more, 10 or more, 25 or more, 50 or more, 100 or more, 200 or more, 300 or more, 400 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, 1000 or more, or 1100 or more oligonucleotides.
- unwanted library fragments comprise those prepared from unwanted RNA sequences.
- library fragments that hybridize to immobilized oligonucleotides comprise library fragments prepared from unwanted RNA sequences.
- the unwanted RNA sequences comprise rRNA.
- the collected library fragments comprise a library depleted of unwanted library fragments.
- the collected library fragments are collected in a reservoir comprised in a sequencer comprising the flowcell. Collected library fragments can then be removed from the reservoir, and the user can perform any additional steps of interest, such as quantification, amplification, quality control, or sequencing.
- all unwanted sequences have at least 90%, at least 95%, or at least 99% homology to a high-abundance RNA sequence in a sample used to prepare the library of fragments. In some embodiments, all unwanted sequences have at least 90%, at least 95%, or at least 99% homology to a high-abundance RNA sequence in a sample used to prepare the library of fragments.
- the library depleted of unwanted library fragments comprises fewer library fragments prepared from unwanted RNA sequences, as compared to the same library before it was added to the solid support.
- the present method of depleting may decrease the number of library fragments prepared from unwanted RNA sequences that are comprised in the collected library.
- a method of depleting further comprises a step of denaturing one or more nucleic acid bound to an immobilized oligonucleotide.
- a method further comprises denaturing library fragments hybridized to immobilized oligonucleotides.
- the denatured library fragments are unwanted library fragments.
- unwanted library fragments are denatured from immobilized oligonucleotides, and unwanted library fragments are siphoned to a waste container.
- a method further comprises denaturing adapter complements hybridized to immobilized oligonucleotides.
- adapter complements are denatured from immobilized oligonucleotides, and adapter complements are siphoned to a waste container.
- a single step denatures both adapter complements and unwanted library fragments.
- both adapter complements and unwanted library fragments are siphoned to a waste container.
- the denaturing is performed with a denaturing agent or heat.
- the denaturing agent is NaOH.
- a method comprises repeating steps.
- the steps of adding a sample, collecting, and denaturing are repeated, wherein the collected library fragments are added back to the solid support after the denaturing.
- multiple rounds of depleting of unwanted library fragments by binding of unwanted library fragments to immobilized oligonucleotides can be performed. Multiple rounds of depleting may increase the percentage of unwanted fragments that are depleted from a library.
- a method further comprises adding the collected library fragments to the solid support after denaturing the hybridized library fragments and/or adapter complements.
- a method of depleting is for depleting library fragments prepared from host RNA.
- host RNA are unwanted RNA sequences, while non-host RNA are desired RNA sequences.
- the unwanted library fragments that hybridize to immobilized oligonucleotides comprise library fragments prepared from host RNA comprised in a sample comprising host RNA and non-host nucleic RNA.
- the depleting method may be for depleting library fragments prepared from host RNA from a sample that comprises both library fragments prepared from host RNA and library fragments prepared from non-host RNA.
- Representative samples that could comprise host RNA and non-host RNA (and be used for library preparation) include samples for assessing a patient’s microbiome or assessing fluids from a patient for an infectious organism (such as a virus, fungus, or bacterium).
- the non-host RNA is microbial.
- the microbe is a bacterium, a virus, and/or a fungus.
- the microbe is a pathogen.
- the microbe is an organism in the host microbiome. In some embodiments, the host is human.
- a method of enriching desired cDNA library fragments from a library of cDNA fragments prepared from RNA comprises (a) preparing a solid support comprising at least one immobilized oligonucleotide, wherein each immobilized oligonucleotide comprises a nucleic acid sequence corresponding to a desired RNA sequence or its complement, (b) adding the library of fragments to the solid support and hybridizing the library fragments to the at least one immobilized oligonucleotide to allow binding of desired library fragments to the at least one immobilized oligonucleotide, and (c) collecting library fragments bound to the at least one immobilized oligonucleotide.
- the desired library fragments comprise those prepared from desired RNA sequences.
- the solid support for enriching comprises a pool of oligonucleotides.
- the pool of oligonucleotides comprises 2 or more, 5 or more, 10 or more, 25 or more, 50 or more, 100 or more, 200 or more, 300 or more, 400 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, 1000 or more, or 1100 or more oligonucleotides.
- the desired RNA sequence has homology to an RNA sequence that a user wishes to study, i.e., an RNA sequence of interest.
- at least one desired RNA sequence has at least 90%, at least 95%, or at least 99% homology to an RNA sequence of interest in a sample used to prepare the library of fragments.
- all desired RNA sequences have at least 90%, at least 95%, or at least 99% homology to an RNA sequence of interest in a sample used to prepare the library of fragments.
- at least one desired RNA sequence is an RNA sequence of interest.
- the collected library fragments comprise a library enriched for desired library fragments.
- the library of fragments added to the solid support is prepared from RNA using a stranded method of cDNA preparation.
- the collecting comprises denaturing the library fragments hybridized to the immobilized oligonucleotides and then collecting the library enriched for desired fragments in a reservoir comprised in a sequencer comprising the solid support.
- the library fragments bound to immobilized oligonucleotides may comprise desired library fragments, and these desired library fragments may be denatured and then collected.
- the denaturing is performed with a denaturing agent or heat.
- the denaturing agent is NaOH.
- the steps of adding the library, denaturing, and collecting library fragments not bound to the solid support are repeated, wherein the collected library fragments not bound to the solid support are then added back to the solid support after the denaturing. Multiple rounds of these steps may lead to greater enrichment of desired library fragments, as more unwanted library fragments may be removed.
- the library enriched for desired library fragments comprises a greater percentage of library fragments prepared from desired RNA sequences, as compared to the library before adding to the solid support. This enrichment can be due to the removal of unwanted library fragments that do not bind to immobilized oligonucleotides comprising desired RNA sequences.
- Additional steps may be performed once an enriched library is prepared (i.e. , bound desired library fragments are denatured and collected).
- the library enriched for desired library fragments is assessed for library size and/or concentration.
- the library enriched for desired library fragments is sequenced.
- the method further comprises amplifying the library enriched for desired library fragments before sequencing.
- the present methods are not limited to a specific type of sample comprising RNA, and these methods can be used with libraries prepared from any sample comprising RNA. Described below are a few exemplary types of samples comprising RNA, wherein sequencing of library fragments prepared from this RNA can be improved by enriching or depleting.
- the sample comprises a microbe sample, a microbiome sample, a bacteria sample, a yeast sample, a plant sample, an animal sample, a patient sample, an epidemiology sample, an environmental sample, a soil sample, a water sample, a metatranscriptomics sample, or a combination thereof.
- the sample comprises an organism of a species that is not predetermined, an unknown species, or a combination thereof.
- a species not predetermined means that a user has not already characterized a given species to be present in the sample.
- the spectrum of bacterial species present in a sample from, for example, soil or gut microbiome may not be predetermined, although the bacterial species later determined to be in the sample may be generally known in the art.
- unknown species refers to a species that has not been previously characterized.
- the sample comprises organisms of at least two species.
- RNA samples refer to samples for generating culturable and non-culturable microbial transcriptome information by large- scale, high-throughput sequencing of transcripts from all microbial communities in specific environmental samples. Metatranscriptomic sequencing allows a user to randomly sequence RNA for understanding complex microbial communities. Methods that can avoid culturing of microbes can allow for data that avoids bias introduced by methods related to individual bacterial isolation and culture.
- the metatranscriptomic sample is a “microbiome sample” from a patient.
- a microbiome sample refers to microorganisms that are present in one or more part of the patient’s body.
- the patient is human.
- the microbiome sample is oral, vaginal, or from the gut.
- the sample from the gut is a stool sample.
- the oral sample is a sample from the tongue.
- the patient is at least 12 months of age, at least 15 months of age, at least 24 months of age, or at least 36 months of age.
- the microbiome sample comprises at least one unwanted RNA molecule from Faecalibacterium, Lachnospiraceae, and/or Clostridium.
- the microbiome sample is vaginal and comprises at least one unwanted RNA molecule from Gardnerella, Lactobacillus, and/or Olsenella.
- the microbiome sample is from tongue and comprises at least one unwanted RNA molecule from Veillonella, Rothia, Streptococcus and/or Prevotella.
- the spectrum of bacterial species present in a sample from, for example, soil or gut microbiome may not be predetermined. Further, bacteria species present in a sample can involve hundreds or perhaps thousands of different species. Consequently, depletion protocols designed against only two representative bacterial species can be insufficient for the needs of the metatranscriptome field. Methods described herein can be used with designing of immobilized oligonucleotides for depleting abundant sequences (e.g., abundant transcripts, such as rRNAs and globin mRNAs) from a sample, such as a complex sample including a metatranscriptomic biosample.
- abundant transcripts e.g., abundant transcripts, such as rRNAs and globin mRNAs
- Metatranscriptomic analysis has a number of applications.
- a user wants to evaluate the microbial population in a patient, as specific bacteria comprised in the patient’s microbiome are linked to either positive or negative effects on the patient.
- a user might want to evaluate the microbiome of a patient exhibiting symptoms of an overactive immune response.
- a user may wish to evaluate the impact of a treatment on a patient’s microbiome using metatranscriptomic analysis.
- Metatranscriptomic samples may comprise a broad spectrum of organisms.
- immobilized oligonucleotides for use in the present methods are designed in an unbiased fashion. In other words, the present methods can be used to prepare enriched libraries from a broad spectrum of organisms, including those which may not be identified, without biasing the library towards known organisms.
- the present methods may be used to deplete known sequences from a metatranscriptomic sample (in which case known sequences would be the unwanted RNA sequences) to prepare a library with a greater percentage of library fragments from unknown sequences.
- known sequences would be the unwanted RNA sequences
- the user could sequence these library fragments at greater depth.
- the sample comprises an organism of a species that is not predetermined, an unknown or unidentified species, or a combination thereof.
- the sample comprises organisms of, of about, of at least, or of at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or a range between any two of these values, species.
- the one or more abundant RNA transcripts can comprise RNA transcripts from organisms of, of about, of at least, or of at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or a range between any two of these values, species.
- the sample can comprise, comprise about, comprise at least, or comprise at most, 1 ng, 2 ng, 3 ng, 4 ng, 5 ng, 6 ng, 7 ng, 8 ng, 9 ng, 10 ng, 20 ng, 30 ng, 40 ng, 50 ng, 60 ng, 70 ng, 80 ng, 90 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, or 1000 ng of RNA transcripts.
- samples may be from a cancer patient (i.e. , an oncology sample).
- oncology samples may be used to evaluate changes in RNA expression in tumor cells, and to potentially monitor these changes over time or over the course of a therapeutic treatment.
- RNA related to tumor markers may be desired RNA.
- RNA from known tumor markers may be used as desired RNA to design oligonucleotides for immobilizing to a solid support for enriching library fragments related to cancer markers.
- oncology samples may be depleted of rRNA and/or mRNA related to other “housekeeping” genes that are not implicated in tumor genesis or progression.
- unwanted RNA can function as carrier nucleic acid. In some embodiments, unwanted RNA serves as carrier molecules for other library fragments. In some embodiments, unwanted RNA serves as carrier molecules for desired library fragments.
- the yield of library fragments after depletion of unwanted library fragments via the present method is greater than the yield of library fragments after depletion of unwanted RNA followed by library preparation in prior art methods.
- sequencing results after library preparation and depletion with the present method may be improved as compared to sequencing results with prior art methods (with upstream depletion of unwanted RNA before library preparation), when aliquots of the same sample comprising RNA is used for the present and prior art methods.
- starting RNA refers to the RNA present in a biological sample, before methods of depletion and library preparation.
- the present methods yield sequencable libraries after depletion when the starting sample comprises less than 100 ng of RNA.
- starting samples comprise less than 100 ng of RNA, less than 50 ng of RNA, less than 20 ng of RNA, less than 10 ng of RNA, or less than 1 ng of RNA.
- a variety of methods are known in the art that allow sequencing data to identify the mRNA strand that was the origin of a library fragment. Use of such “stranded” methods can allow the user to determine the sequence of the original mRNA strand using the sequence of the first strand of cDNA (without confounding data from a second strand of cDNA).
- a library of fragments added to the solid support is prepared from RNA using a stranded method of cDNA preparation.
- a stranded method of cDNA preparation means that most library fragments after an amplification step will correspond to the complementary sequence of an undesired RNA. In this way, unwanted fragments after amplification can generally be depleted by immobilized oligonucleotides corresponding to the undesired RNA.
- a user may prefer to use a non-stranded method of cDNA preparation.
- cDNA is prepared by a non-stranded method and a user wants to deplete unwanted RNA
- the user may prefer to immobilize oligonucleotides corresponding to both the unwanted RNA sequence and its complement to increase efficiency of the depleting.
- cDNA is prepared by a non-stranded method and a user wants to enrich desired RNA
- the user may prefer to immobilize oligonucleotides corresponding to both the desired RNA sequence and its complement to increase efficiency of the enriching.
- An exemplary method of stranded cDNA preparation is outlined in “TruSeq Stranded Total RNA Reference Guide,” Illumina, 2017.
- the mRNA is copied into a first strand of cDNA using reverse transcriptase in a First Strand Synthesis Actinomycin Mix, which allows RNA-dependent synthesis and prevents undesired DNA-dependent synthesis.
- the First Strand Synthesis Actinomycin Mix can improve strand specificity when generating a first strand of cDNA.
- Second strand cDNA synthesis is performed using DNA polymerase I and RNase H in a Second Strand Marking Mix, wherein dTTP has been replaced by dUTP. Incorporation of dUTP in the second strand of cDNA can quench amplification of this strand when a uracil-intolerant DNA polymerase is used.
- the nucleoside trisphosphates comprised in a composition for first strand cDNA synthesis comprises dCTP, dATP, dGTP, and dTTP.
- dTTP is replaced with dUTP in a second strand cDNA synthesis reaction for strand specificity.
- a composition for second strand cDNA synthesis comprises dCTP, dATP, dGTP, and dUTP.
- incorporation of dUTP in the second strand of cDNA suppresses amplification of the second strand of cDNA in the index PCR reaction during library preparation.
- suppression of amplification of the second strand of cDNA allows for strandspecific methods.
- a uracil-intolerant DNA polymerase may be used in stranded methods of cDNA preparation comprising amplification.
- the presence of uracil in a second strand of cDNA prepared from RNA in a sample can quench amplification of this second strand when a uracil-intolerant DNA polymerase is used. In this way, the amplified cDNA is limited to that generated from the first strand of cDNA from an RNA that was comprised in the sample.
- cDNA preparation is by a non-stranded method that does retain strand information from the mRNA.
- Libraries prepared by any method can be used together with the present methods of enriching or depleting.
- a method of library preparation prepares double-stranded library fragments, and the double-stranded library fragments are denatured before being added to a solid support.
- a library fragment may be single-stranded when they are available to hybridize to an immobilized oligonucleotide comprising a sequence all or partially complementary to the library fragment.
- immobilized oligonucleotides are single-stranded to allow for hybridizing and capturing of single-stranded library fragments that are complementary.
- specific binding of a single-stranded library fragment to an immobilized oligonucleotide generates a double-stranded oligonucleotide.
- the immobilized oligonucleotide specifically bound to the library fragment may be bound with a high-enough affinity to avoid denaturing of this double-stranded oligonucleotide in standard washing steps. In this way, library fragments with specific binding to an immobilized oligonucleotide may remain bound during washing steps and removal of unbound library fragments.
- one or more adapter sequence are incorporated into library fragments.
- Such adapter sequences comprised in library fragments may be termed “library adapters.”
- a given library adapter sequence may universal, meaning that all or most library fragments comprise this library adapter sequence.
- library adapter sequences are incorporated into library fragments during library preparation. In some embodiments, library adapter sequences are incorporated into library fragments after methods of depleting or enriching as described herein.
- Adapter sequences can be any known in the art, and one skilled in the art can choose adapter sequences based on any downstream method (such as sequencing) and what platform will be used for the downstream method (such as a particular sequencer). Further, a library adapter sequence can be designed to bind to a solid support adapter sequence comprised in an immobilized oligonucleotide on a solid support.
- a library fragment comprises one or more adapter sequence in addition to the library adapter sequence for binding to the solid support adapter.
- an adapter sequence comprises a primer sequence, an index tag sequence, a capture sequence, a barcode sequence, a cleavage sequence, or a sequencing- related sequence, or a combination thereof.
- a sequencing-related sequence may be any sequence related to a later sequencing step.
- a sequencing-related sequence may work to simplify downstream sequencing steps.
- a sequencing-related sequence may be a sequence that would otherwise be incorporated via a step of ligating an adapter to nucleic acid fragments.
- the adapter sequence comprises a P5 (SEQ ID NO: 1132) or P7 sequence (SEQ ID NO: 1133), and/or their complement, to facilitate binding to a flowcell in certain sequencing methods.
- P5 SEQ ID NO: 1132
- P7 sequence SEQ ID NO: 1133
- an adapter comprises a region for cluster amplification. In some embodiments, an adapter comprises a region for priming a sequencing reaction.
- an adapter comprises an A14 primer binding sequence (SEQ ID NO: 1134). In some embodiments, an adapter comprises a B15 primer binding sequence (SEQ ID NO: 1135).
- methods described herein comprise one or more amplification step.
- library fragments are amplified before being added to a solid support.
- library fragments are amplified after a method of enriching or depleting described herein.
- amplifying is by PCR amplification.
- library fragments are amplified before being added to a solid support.
- amplifying of library fragments is comprised in a method of library preparation.
- amplification with a uracil-intolerant DNA polymerase is used to selectively amplify cDNA strands prepared as a first strand from RNA (without amplifying second strands of DNA that comprise uracil).
- the library fragments added to the solid support may comprise mostly fragments comprising a sequence complementary to a desired RNA or unwanted RNA.
- the library fragments may comprise mostly fragments prepared from a first strand of cDNA.
- more than 70%, more than 80%, more than 90%, or more than 95% of library fragments comprise cDNA from a first strand of cDNA.
- collected library fragments are amplified after a method of depleting or enriching.
- a depleted library is amplified.
- an enriched library is amplified.
- the amplifying is performed with a thermocycler. In some embodiments, the amplifying is by PCR amplification.
- the amplifying is performed without PCR amplification. In some embodiments, the amplifying does not require a thermocycler. In some embodiments, enriching/depleting and amplifying after the enriching/depleting is performed in a sequencer.
- the amplifying is performed without a thermocycler.
- the amplifying is performed by bridge or cluster amplification.
- library fragments comprising library adapter sequences can bind to immobilized oligonucleotides comprising solid support adapter sequences. This binding can allow for standard bridge amplification.
- bridge amplification is performed on the same solid support used for enriching or depleting.
- bridge amplification is performed after adding the collected library fragments to the solid support and allowing the library adapters comprised in the collected library fragments to bind to the solid support adapter sequences, wherein the adding is performed after denaturing the hybridized library fragments and/or adapter complements.
- a method of amplifying desired cDNA library fragments from a library of cDNA fragments prepared from RNA comprises: a. providing a solid support having two pools of immobilized oligonucleotides on its surface, wherein the first pool comprises immobilized oligonucleotides each comprising an unwanted RNA sequence and the second pool comprises immobilized oligonucleotides each comprising a solid support adapter sequence that can bind to a library adapter comprised in library fragments, wherein adapter complements are reversibly bound to the solid support adapter sequences, b.
- the immobilized DNA fragments can be amplified using cluster amplification methodologies as exemplified by the disclosures of US Patent Nos. 7,985,565 and 7,115,400, the contents of each of which is incorporated herein by reference in its entirety.
- the incorporated materials of US Patent Nos. 7,985,565 and 7,115,400 describe methods of solid-phase nucleic acid amplification which allow amplification products to be immobilized on a solid support in order to form arrays comprised of clusters or “colonies” of immobilized nucleic acid molecules.
- Each cluster or colony on such an array is formed from a plurality of identical immobilized polynucleotide strands and a plurality of identical immobilized complementary polynucleotide strands.
- the arrays so-formed are generally referred to herein as “clustered arrays.”
- the products of solidphase amplification reactions such as those described in US Patent Nos. 7,985,565 and 7,115,400 are so-called “bridged” structures formed by annealing of pairs of immobilized polynucleotide strands and immobilized complementary strands, both strands being immobilized on the solid support at the 5’ end, in some embodiments via a covalent attachment.
- Cluster amplification methodologies are examples of methods wherein an immobilized library fragment is used to produce immobilized amplicons.
- a library depleted of unwanted library fragments is sequenced.
- a library enriched for desired library fragments is sequenced.
- the collected library may comprise less than 15%, 13%, 11%, 9%, 7%, 5%, 3%, 2% or 1% or any range in between of unwanted RNA species.
- the collected library after enriching or depleting comprises at least 99%, 98%, 97%, 95%, 93%, 91%, 89% or 87% or any range in between of desired RNA.
- the library for sequencing after the enriching or depleting mainly comprises library fragments that were prepared from RNA of interest.
- sequencing data generated after depleting of unwanted library fragments has fewer sequences corresponding to unwanted RNA as compared to the same library sequenced without the depleting.
- sequencing data generated after enriching of desired library fragments has a higher percentage of sequences corresponding to desired RNA as compared to the same library sequenced without the enriching.
- Depleted or enriched libraries prepared by the present method can be used with any type of RNA sequencing, such as RNA-seq, small RNA sequencing, long noncoding RNA (IncRNA) sequencing, circular RNA (circRNA) sequencing, targeted RNA sequencing, exosomal RNA sequencing, and degradome sequencing.
- RNA sequencing such as RNA-seq, small RNA sequencing, long noncoding RNA (IncRNA) sequencing, circular RNA (circRNA) sequencing, targeted RNA sequencing, exosomal RNA sequencing, and degradome sequencing.
- RNA sequencing a user may prepare by depleted of linear RNA with digestion of linear RNA, followed by library preparation and depleting of rRNA by a method described herein. As such, the present methods can easily be combined with other steps in known protocols related to RNA sequencing.
- Depleted or enriched libraries can be sequenced according to any suitable sequencing methodology, such as direct sequencing, including sequencing by synthesis, sequencing by ligation, sequencing by hybridization, nanopore sequencing and the like.
- the depleted or enriched libraries are sequenced on a solid support.
- the solid support for sequencing is the same solid support on which the enriching or depleting is performed.
- the solid support for sequencing is the same solid support upon which amplification occurs after the enriching or depleting.
- Flowcells provide a convenient solid support for performing sequencing.
- One or more library fragments (or amplicons produced from library fragments) in such a format can be subjected to an SBS or other detection technique that involves repeated delivery of reagents in cycles.
- SBS SBS
- one or more labeled nucleotides, DNA polymerase, etc. can be flowed into/through a flowcell that houses one or more amplified nucleic acid molecules. Those sites where primer extension causes a labeled nucleotide to be incorporated can be detected.
- the nucleotides can further include a reversible termination property that terminates further primer extension once a nucleotide has been added to a primer.
- a nucleotide analog having a reversible terminator moiety can be added to a primer such that subsequent extension cannot occur until a deblocking agent is delivered to remove the moiety.
- a deblocking reagent can be delivered to the flowcell (before or after detection occurs). Washes can be carried out between the various delivery steps. The cycle can then be repeated n times to extend the primer by n nucleotides, thereby detecting a sequence of length n.
- Performing sequencing, and optionally performing amplifying, on the same solid support used for the depleting and/or enriching can reduce the number of hands-on steps for the user and sample loss that would be associated with transferring sample from one solid support to another.
- RNA sequencing is often times difficult due to an abundance of unwanted transcripts such as ribosomal RNA (rRNA) that can dominate a sample and swamp out the RNA sequences of interest. If the unwanted transcripts are not removed, analysis of the transcriptome could be compromised. Therefore, depleting unwanted RNA from a microbiome sample comprising nucleic acid prior to analysis such as sequencing or other downstream applications can increase the specificity and accuracy of the desired analysis. Exemplary methods of depleting rRNA are described in WO 2020132304 Al, which is incorporated herein in its entirety.
- rRNA ribosomal RNA
- the present disclosure describes methods and materials useful in depleting rRNA species from a nucleic acid sample such that the RNA of importance can be studied and is not lost in the sea of undesired RNA transcripts.
- the nucleic acid sample may be any described herein, such as a metatranscriptomic sample.
- a microbiome sample may contain RNA or DNA or both, including both undesired (off-target or unwanted) and desired (target) nucleic acids.
- the DNA or RNA in the sample can be either unmodified or modified and includes, but is not limited to, single or double stranded DNA or RNA or derivatives thereof (e.g., some regions of the DNA or RNA are double stranded whereas concurrently other regions of the DNA or RNA are single stranded) and the like.
- a microbiome sample may also contain cells from the host.
- a gut microbiome patient from a human patient i.e., the “host” may comprise microorganisms present in the gut as well as host cells, such that the sample comprises nucleic acids from both the host and microorganisms.
- a microbiome sample may include any chemically, enzymatically, and/or metabolically modified forms of nucleic acids as well as any unmodified forms of nucleic acids, or combinations thereof.
- a microbiome sample can contain both wanted and unwanted nucleic acids.
- Unwanted nucleic acids include those nucleic acids from the host as well as rRNA from microorganisms.
- Wanted or desired nucleic acids are those nucleic acids that are the basis or focus of study, the target nucleic acids.
- RNA from microorganisms would be considered unwanted nucleic acids and other RNA from microorganisms is the target nucleic acid.
- unwanted RNA is rRNA.
- a microbiome sample could contain the desired RNA (such as mRNA) from microorganisms while also including undesired rRNA.
- RNA extraction from a gross sample like blood, tissue, cells, fixed tissues, etc., are well known in the art, as found in Current Protocols for Molecular Biology (John Wiley & Sons) and multitude molecular biology methods manuals.
- RNA isolation can be performed by commercially available purification kits, for example Qiagen RNeasy minicolumns, MasterPure Complete DNA and RNA Purification Kits (Epicentre), Parrafin Block RNA Isolation Kit (Ambion), RNA-Stat-60 (Tel-Test) or cesium chloride density gradient centrifugation.
- the current methods are not limited by how the RNA is isolated from a sample prior to RNA depletion.
- methods include use of probes to host unwanted RNA and/or microbial unwanted RNA.
- methods described herein may include the use of probes directed to non-microbial RNA (such as the DPI probe set described herein) as well as probes directed to microbial rRNA (such as HMvl and/or HMv2 probe sets described herein), as described in Example 5.
- a method for depleting unwanted RNA molecules comprised in a patient microbiome sample wherein the patient microbiome sample comprises at least one target RNA or DNA sequence and at least one unwanted RNA molecule, comprises
- RNA sequencing a plurality of probe-development microbiome samples to determine at least one unwanted RNA molecule comprising a bacterial ribosomal RNA (rRNA) sequence from sequencing data (b) preparing a probe set comprising at least one DNA probe complementary to the at least one unwanted RNA molecule; (c) contacting the patient microbiome sample with the probe set to prepare DNA:RNA hybrids; and (d) contacting the DNA:RNA hybrids with a ribonuclease that degrades the RNA from the DNA:RNA hybrids, thereby degrading the unwanted RNA molecules in the patient microbiome sample to form a degraded mixture.
- rRNA bacterial ribosomal RNA
- a method for depleting unwanted RNA molecules comprised in a patient microbiome sample wherein the patient microbiome sample comprises at least one target RNA or DNA sequence and at least one unwanted RNA molecule, comprises
- a method further comprises (a) degrading any remaining DNA probes by contacting the degraded mixture with a DNA digesting enzyme, optionally wherein the DNA digesting enzyme is DNase I, to form a DNA degraded mixture; and
- the addition of a destabilizer such as formamide helps remove some unwanted RNA that was shown to be more problematic to deplete if formamide was not present.
- formamide may serve to relax structural barriers in the unwanted RNA (such as rRNA) so that the DNA probes can bind more efficiently.
- the addition of formamide has demonstrated the added benefit of improving the detection of some non-targeted transcripts possibly by denaturing/r el axing regions of the RNAs, for example, which have very stable secondary or tertiary structures and are not normally well represented well in other library preparation methods.
- the contacting with the probe set comprises treating the nucleic acid sample with a destabilizer.
- the destabilizer is heat and/or a nucleic acid destabilizing chemical.
- the nucleic acid destabilizing chemical comprises betaine, DMSO, formamide, glycerol, or a derivative thereof, or a mixture thereof.
- the nucleic acid destabilizing chemical comprises formamide.
- the formamide is present during the contacting with the probe set at a concentration of from about 10 to 45% by volume.
- treating the sample with heat comprises applying heat above the melting temperature of the at least one DNA:RNA hybrid.
- the ribonuclease is RNase H or hybridase.
- the unwanted RNA is converted to a DNA:RNA hybrid by hybridizing partially or completely complementary DNA probes to the unwanted RNA molecules.
- Methods for hybridizing nucleic acid probes to nucleic acids are well-established in the sciences and whether a probe is partially or completely complementary with the partner sequence, the fact that a DNA probe hybridizes to the unwanted RNA species following washes and other manipulations of the sample demonstrates a DNA probe that can be used in methods of the present disclosure.
- DNA can also be considered an unwanted nucleic acid if the target for study is an RNA, at which point DNA can also be removed by depletion.
- an RNA sample is denatured in the presence of DNA probes.
- the DNA probes are added to the denatured RNA sample (denatured at 95 °C for 2 min.) whereupon cooling the reaction to 37 °C for 15-30 minutes results in hybridization of the DNA probes to their respective target RNA sequences thereby creating DNA:RNA hybrid molecules.
- contacting with the probe set comprises treating the nucleic acid sample with a destabilizer.
- a destabilizer is heat or a nucleic acid destabilizing chemical.
- a nucleic acid destabilizing chemical is betaine, DMSO, formamide, glycerol, or a derivative thereof, or a mixture thereof.
- a nucleic acid destabilizing chemical is formamide or a derivative thereof, optionally wherein the formamide or derivative thereof is present at a concentration of from about 10 to 45% of the total hybridization reaction volume.
- treating the sample with heat comprises applying heat above the melting temperature of the at least one DNA: RNA hybrid.
- formamide is added to the hybridization reaction regardless of RNA sample source (e.g., human, mouse, rat, etc.).
- RNA sample source e.g., human, mouse, rat, etc.
- hybridizing to the DNA probes is performed in the presence of at least 3%, 5%, 10%, 20%, 25%, 30%, 35%, 40%, or 45% by volume of formamide.
- a hybridization reaction for RNA depletion includes approximately 25% to 45% by volume of formamide.
- a ribonuclease that degrades RNA from a DNA: RNA hybrid may be added to the reaction.
- a ribonuclease is RNase H or Hybridase.
- RNase H (NEB) or Hybridase (Lucigen) are examples of enzymes that will degrade RNA from a DNA: RNA hybrid.
- Degradation by a ribonuclease such as RNase H or Hybridase degrades the RNA into small molecules that can then be removed.
- RNase H is reported to digest RNA from a DNA:RNA hybrid approximately every 7-21 bases (Schultz et al., J. Biol. Chem. 2006, 281:1943-1955; Champoux and Schultz, FEBS J. 2009, 276:1506-1516).
- the digestion of the RNA of the DNA:RNA hybrid can occur at 37 °C for approximately 30 minutes.
- the methods comprise contacting the ribonucleasedegraded mixture with a DNA digesting enzyme, thereby degrading DNA in the mixture.
- the digested sample is exposed to a DNA digesting enzyme such as DNase I, which degrades the DNA probes.
- the DNase DNA digestion reaction is incubated, for example, at 37 °C for 30 minutes, after which point the DNase enzyme can be denatured at 75 °C for a period of time as necessary to denature the DNase, for example for up to 20 minutes.
- the depletion method comprises separating the degraded RNA from the degraded mixture.
- separating comprises purifying the target RNA from the degraded RNA (and degraded DNA if present), for example, using a nucleic acid purification medium, such as RNA capture beads, such as RNAClean XP beads (Beckman Coulter).
- RNA capture beads such as RNAClean XP beads (Beckman Coulter).
- RNAClean XP beads Beckman Coulter.
- the target RNA can be enriched by removing the degraded products while leaving the desired and longer RNA targets behind. Suitable enrichment methods include treating the degraded mixture with magnetic beads which bind to the desired fragment size of the enriched RNA targets, spin columns, and the like.
- magnetic beads such as AMPure XP beads, SPRISelect beads, RNAClean XP beads (Beckman Coulter) can be used, as long as the beads are free of RNases (e.g., Quality Controlled to be RNase free).
- RNases e.g., Quality Controlled to be RNase free.
- RNAClean XP beads target 100 nucleotides or longer nucleic acid fragments
- SPRISelect beads target 150 to 800 nucleotide nucleic acid fragments and do not target shorter nucleic acid sequences such as the degraded RNA and DNA that results from the enzymatic digestions of RNase H and DNase.
- the mRNA can be further enriched by capture using, for example, beads that comprise oligodT sequences for capturing the mRNA adenylated tails. Methods of mRNA capture are well-known by skilled artisans.
- the target RNA has been purified away from the reaction components including the undesired degraded nucleic acids, additional sample manipulation can occur.
- the enriched target total RNA followed by an exemplary library preparation workflow that is typical for subsequent sequencing on, for example, an Illumina sequencer.
- these workflows are exemplary only and a skilled artisan will understand that the enriched RNA can be used in multitude additional applications such as PCR, qPCR, microarray analysis, and the like either directly or following additional manipulation such as converting the RNA to cDNA by using established and will understood protocols.
- RNA depletion may result in a sample enriched with the target RNA molecules.
- the methods described herein result is a depleted RNA sample comprising less than 15%, 13%, 11%, 9%, 7%, 5%, 3%, 2% or 1% or any range in between of the unwanted RNA species.
- the enriched RNA sample then comprises at least 99%, 98%, 97%, 95%, 93%, 91%, 89% or 87% or any range in between of the target total RNA.
- the DNA probes do not hybridize to the entire contiguous length of an RNA species to be deleted.
- the full-length sequence of a RNA species targeted for depletion need not be targeted with a full-length DNA probe, or a probe set that tiles contiguously over the entire RNA sequence.
- DNA probes described herein leave gaps such that the DNA: RNA hybrids formed are not contiguous.
- gaps of at least 5 nucleotides, 10 nucleotides, 15 nucleotides or 20 nucleotides between DNA: RNA hybrids provided efficient RNA depletion.
- probe sets that include gaps can hybridize more efficiently to the unwanted RNA, as the DNA probes do not hinder hybridization of adjacent probes as could potentially occur with probes that cover the whole RNA sequence targeted for depletion, or probes that overlap one another.
- the at least one DNA probe comprise 2 or more, 5 or more, 10 or more, 25 or more, 50 or more, 100 or more, 200 or more, 300 or more, 400 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, 1000 or more, 1100 or more, or 1131 sequences selected from SEQ ID NOs: 1-1131 or its complement.
- the at least one DNA probe comprises 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, 1000 or more, 1100 or more, or 1131 sequences selected from SEQ ID NOs: 1-1131 or its complement.
- the at least one DNA probe comprises at least one HMvl sequence and comprising at least one of SEQ ID NOs: 1-10, 12-18, 21, 22, 24-33, 35, 39-43, 45-48, 50-73, 75, 77, 78, 81-84, 86-103, 105-107, 109-127, 129-134, 136-140, 142, 143, 148, 149, 151-155, 157, 160-165, 168-174, 176-182, 184-187, 189-194, 196-204, 206, 208-215, 217-223, 225, 227-246, 248-265, 269, 270, 272-277, 279, 281, 282, 284-290, 292-301, 303-321, 323-331, 333-336, 338, 340-342, 344, 346-349, 351-355, 357-359, 361- 372, 374-380, 383-386, 388, 3
- the at least one DNA probe comprises 100 or more, 500 or more, or 1000 or more sequences comprising at least one of SEQ ID NOs: 1-10, 12-18, 21, 22, 24-33, 35, 39-43, 45-48, 50-73, 75, 77, 78, 81-84, 86-103, 105-107, 109-127, 129-134, 136-140, 142, 143, 148, 149, 151-155, 157, 160-165, 168-174, 176-182, 184-187, 189-194, 196-204, 206, 208-215, 217-223, 225, 227-246, 248-265, 269, 270, 272-277, 279, 281, 282, 284-290, 292-301, 303-321, 323-331, 333-336, 338, 340-342, 344, 346-349, 351- 355, 357-359, 361-372, 374-380, 383-386
- the at least one DNA probe comprises a sequence comprising each of SEQ ID NOs: 1-10, 12-18, 21, 22, 24-33, 35, 39-43, 45-48, 50- 73, 75, 77, 78, 81-84, 86-103, 105-107, 109-127, 129-134, 136-140, 142, 143, 148, 149, 151- 155, 157, 160-165, 168-174, 176-182, 184-187, 189-194, 196-204, 206, 208-215, 217-223, 225, 227-246, 248-265, 269, 270, 272-277, 279, 281, 282, 284-290, 292-301, 303-321, 323- 331, 333-336, 338, 340-342, 344, 346-349, 351-355, 357-359, 361-372, 374-380, 383-386, 388, 390, 391, 393, 3
- the at least one DNA probe further comprises at least one sequence of the HMv2 sequences and comprising at least one of SEQ ID NOs: 19, 74, 76, 85, 104, 108, 158, 175, 226, 278, 322, 339, 389, 513, 517, 520, 546, 553, 609, 611, 650, 662, 677, 683, 686, 706, 780, 827, 835, 882, 1022, 1059, 1077, 1078, 1098, 1106, 1111, 1114, 1128, and 1131.
- the at least one DNA probe comprises 10 or more, 20 or more, or 30 or more sequences comprising at least one of SEQ ID NOs: 19, 74, 76, 85, 104, 108, 158, 175, 226, 278, 322, 339, 389, 513, 517, 520, 546, 553, 609, 611, 650, 662, 677, 683, 686, 706, 780, 827, 835, 882, 1022, 1059, 1077, 1078, 1098, 1106, 1111, 1114, 1128, and 1131.
- the at least one DNA probe comprises a sequence comprising each of SEQ ID NOs: 19, 74, 76, 85, 104, 108, 158, 175, 226, 278, 322, 339, 389, 513, 517, 520, 546, 553, 609, 611, 650, 662, 677, 683, 686, 706, 780, 827, 835, 882, 1022, 1059, 1077, 1078, 1098, 1106, 1111, 1114, 1128, and 1131.
- the at least one DNA probe comprises at least one HMvl sequence or HMv2 sequence and comprising at least one of SEQ ID NOs: 1-10, 12-19, 21, 22, 24-33, 35, 39-43, 45-48, 50-78, 81-127, 129-134, 136-140, 142, 143, 148, 149, 151-155, 157, 158, 160-165, 168-182, 184-187, 189-194, 196-204, 206, 208-215, 217-223, 225-246, 248-265, 269, 270, 272-279, 281, 282, 284-290, 292-301, 303-331, 333-336, 338- 342, 344, 346-349, 351-355, 357-359, 361-372, 374-380, 383-386, 388-391, 393, 395-401, 403-406, 408-420, 422-439, 441, 443, 444, 446-460, 462-4
- the at least one DNA probe comprises 100 or more, 500 or more, or 1000 or more sequences comprising at least one of SEQ ID NOs: 1-10, 12-19, 21, 22, 24-33, 35, 39-43, 45-48, 50-78, 81-127, 129-134, 136-140, 142, 143, 148, 149, 151-155, 157, 158, 160-165, 168-182, 184-187, 189-194, 196-204, 206, 208-215, 217-223, 225-246, 248-265, 269, 270, 272-279, 281, 282, 284-290, 292-301, 303-331, 333-336, 338- 342, 344, 346-349, 351-355, 357-359, 361-372, 374-380, 383-386, 388-391, 393, 395-401, 403-406, 408-420, 422-439, 441, 443, 444, 446-460, 462-466
- the at least one DNA probe comprises a sequence comprising each of SEQ ID NOs: 1-10, 12-19, 21, 22, 24-33, 35, 39-43, 45-48, 50- 78, 81-127, 129-134, 136-140, 142, 143, 148, 149, 151-155, 157, 158, 160-165, 168-182, 184-187, 189-194, 196-204, 206, 208-215, 217-223, 225-246, 248-265, 269, 270, 272-279, 281, 282, 284-290, 292-301, 303-331, 333-336, 338-342, 344, 346-349, 351-355, 357-359, 361-372, 374-380, 383-386, 388-391, 393, 395-401, 403-406, 408-420, 422-439, 441, 443, 444, 446-460, 462-466, 468, 469, 471, 473-4
- the at least one DNA probe further comprises at least one sequence of the DPI sequences and comprising at least one of SEQ ID NOs: 11, 20, 23, 34, 36-38, 44, 49, 79, 80, 128, 135, 141, 144-147, 150, 156, 159, 166, 167, 183, 188, 195, 205, 207, 216, 224, 247, 266-268, 271, 280, 283, 291, 302, 332, 337, 343, 345, 350, 356, 360, 373, 381, 382, 387, 392, 394, 402, 407, 421, 440, 442, 445, 461, 467, 470, 472, 478, 503,
- the at least one DNA probe comprises 10 or more, 20 or more, or 30 or more sequences comprising at least one of SEQ ID NOs: 11, 20, 23, 34, 36-38, 44, 49, 79, 80, 128, 135, 141, 144-147, 150, 156, 159, 166, 167, 183, 188, 195, 205, 207, 216, 224, 247, 266-268, 271, 280, 283, 291, 302, 332, 337, 343, 345, 350, 356, 360, 373, 381, 382, 387, 392, 394, 402, 407, 421, 440, 442, 445, 461, 467,
- the at least one DNA probe comprises a sequence comprising each of SEQ ID NOs: 11, 20, 23, 34, 36-38, 44, 49, 79, 80, 128, 135, 141, 144-147, 150, 156, 159, 166, 167, 183, 188, 195, 205, 207, 216, 224, 247, 266-268, 271, 280, 283, 291, 302, 332, 337, 343, 345, 350, 356, 360, 373, 381, 382, 387, 392, 394, 402, 407, 421, 440, 442, 445, 461, 467, 470, 472, 478, 503, 515, 525, 530,
- the method depletes 70% or greater, 80% or greater, 90% or greater, or 95% or greater of bacterial rRNA comprised in the microbiome sample.
- At least one probe is comprised in a kit or composition.
- the at least one probe may be any combination of probes disclosed herein.
- a composition comprising a probe set comprises at least one DNA probe comprising at least one sequence comprising at least one of SEQ ID NOs: 1-1131; and a ribonuclease capable of degrading RNA in an DNA: RNA hybrid.
- the ribonuclease is RNase H.
- a kit comprising a probe set comprises at least one DNA probe comprising at least one sequence comprising at least one of SEQ ID NOs: 1- 1131; and a ribonuclease capable of degrading RNA in an DNA:RNA hybrid.
- kit comprises a probe set comprising at least one DNA probe comprising at least one of SEQ ID NOs: 1-1131; a ribonuclease; a DNase; and RNA purification beads.
- the ribonuclease is RNase H.
- a kit further comprises an RNA depletion buffer, a probe depletion buffer, and a probe removal buffer.
- a kit further comprises a nucleic acid destabilizing chemical.
- the nucleic acid destabilizing chemical comprises betaine, DMSO, formamide, glycerol, or a derivative thereof, or a mixture thereof.
- the nucleic acid destabilizing chemical comprises formamide.
- a method of rRNA depletion followed by amplification via thermal cycler can be performed.
- This method would utilize current flowcells used for sequencing, featuring inlet ports for the sequence fluidics system to pump buffers and reagents onto the flowcell and to siphon reagents to a waste container.
- oligonucleotide sequences would be tethered (i.e., immobilized) to the surface of the flowcell, and rRNA sequences would be comprised in these immobilized oligonucleotides.
- the user would load RNA libraries (i.e.
- Figure 1 outlines a representative method for depletion. As library molecules flow in solution, library fragments generated from rRNA transcripts will hybridize to complementary sequences tethered to the flowcell while library fragments generated from non-rRNA transcripts will continue to flow unimpeded to a storage chamber for collection. After the hybridization step is complete, the user would discard the flowcell and collect the siphoned non-rRNA library fragments for PCR amplification, cleanup quantification, quality control, and sequencing.
- This method would leverage the advantages of current flowcell/sequencer capabilities for a user-friendly method of depleting unwanted library fragments, such as those library fragments prepared from rRNA.
- Methods can also be designed to deplete library fragments prepared from rRNA and amplify library fragments prepared from non-rRNA on the same solid support.
- This flowcell-like solid support would comprise a pool of immobilized oligonucleotides comprising rRNA sequences.
- the solid support would also comprise another pool of immobilized oligonucleotides comprising double-stranded P5 and/or P7 oligonucleotides immobilized on the surface.
- the double-stranded P5 and/or P7 oligonucleotides would comprise an adapter complement that is an oligonucleotide reversibly bound to the P5 and/or P7 adapter sequence (i. e. , a solid support adapter sequence).
- Library fragments could be prepared by standard methods after cDNA preparation from a sample comprising RNA. These library fragments can be prepared by incorporating library adapter sequences that can bind to P5 and/or P7. Library fragments generated from rRNA transcripts would bind to the surface of the flowcell based on hybridizing to immobilized oligonucleotides comprising rRNA sequences, while library fragments prepared from non-rRNA transcripts would flow unimpeded and be siphoned for temporary storage in a reservoir.
- a denaturing reagent such as NaOH would be pumped across the flowcell device causing the hybridized library fragments prepared from rRNA and the untethered strand of the double-stranded P5 and/or P7 oligonucleotides to dissociate from the flowcell into a waste reservoir. Then the collected library fragments (comprising library fragments prepared from non-rRNA) would be reintroduced to the flowcell from the temporary storage chamber for binding to the single-stranded immobilized oligonucleotides comprising P5 and/or P7.
- bridge amplification chemistry can amplify the library fragments. After bridge amplification has generated enough library fragments, a cleavage step can be done as in current sequencing chemistry to release both the forward and reverse strands for subsequent collection, quantification, and quality control prior to sequencing.
- a solid support such as a flowcell
- a user could prepare oligonucleotides corresponding to desired RNA and immobilize these oligonucleotides to a solid support.
- a user may want to enrich for RNA sequences associated with cancer markers for evaluating treatment response, tumor progression, or other means of evaluation (i.e., desired RNA), and the user can immobilize oligonucleotides comprising sequences from such RNA to a solid support.
- a flowcell with such immobilized oligonucleotides may be termed an enrichment flowcell.
- the user can then prepare a cDNA library as described above in Example 1 from a patient sample comprising RNA.
- Library fragments can then be added to the enrichment flowcell.
- Library fragments prepared from desired RNA would bind to the enrichment flowcell, and the user can siphon fluid that does not bind to the enrichment flowcell (comprising library fragments not prepared from desired RNA) to a waste container.
- the user can then denature the bound library fragments, collect them, and sequence them (with optional amplification before sequencing). In this way, the library that is sequenced will be enriched for library fragments prepared from desired RNA.
- Some human-associated microbiome samples may have significant amounts of host (human) RNA in addition to bacterial RNA (such as rRNA).
- host (human) RNA such as rRNA
- skin, oral, and vaginal sample are expected to have a lot of human cells included, so probes against human sequences and bacterial sequences unwanted sequences together may provide the best results for depleting unwanted sequences from human microbiome samples.
- HMvl probe set also includes probes that were designed directly against the rRNA sequences from all 38 species present in the ATCC mock community samples (MSA-2002, - 2005 & -2006) as well as E. coli and B. subtilis.
- rRNA depletion was less efficient for a subset of donors in the 9- to 15-months old group. Taxonomic analysis revealed that these samples had high levels of Bifidobacterium bifldum. Lack of depletion suggests that the HMvl probe set relatively poorly targets rRNA from this particular species. [00462] Additional probes targeting Bifidobacterium bifidum were designed using the present iterative process and added to the HMvl probe pool to create a second human microbiome pool (HMv2). Further experiments were performed with the HM probes set comprising both HMvl probes and HMv2 probes.
- the RiboZero RNase method with the HM probes improves depletion of rRNA from human microbiome samples as compared to the RiboZero standard method (with DPI probes) or the RiboZero-Bact kit using probe-based hybridization and probes designed for depleting rRNA from E. coli and B. subtilis.
- the HM probes are well-suited for depleting rRNA from human microbiome samples.
- the term about refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated.
- the term about generally refers to a range of numerical values (e.g., +/-5- 10% of the recited range) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result).
- the terms modify all of the values or ranges provided in the list.
- the term about may include numerical values that are rounded to the nearest significant figure.
Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA3223045A CA3223045A1 (en) | 2021-09-30 | 2022-09-29 | Solid supports and methods for depleting and/or enriching library fragments prepared from biosamples |
CN202280042563.XA CN117545852A (en) | 2021-09-30 | 2022-09-29 | Solid support and method for depleting and/or enriching library fragments prepared from biological samples |
AU2022354071A AU2022354071A1 (en) | 2021-09-30 | 2022-09-29 | Solid supports and methods for depleting and/or enriching library fragments prepared from biosamples |
US17/937,021 US20230094911A1 (en) | 2021-09-30 | 2022-09-30 | Solid Supports and Methods for Depleting and/or Enriching Library Fragments Prepared from Biosamples |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US202163250563P | 2021-09-30 | 2021-09-30 | |
US63/250,563 | 2021-09-30 | ||
US202263351170P | 2022-06-10 | 2022-06-10 | |
US63/351,170 | 2022-06-10 |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US17/937,021 Continuation US20230094911A1 (en) | 2021-09-30 | 2022-09-30 | Solid Supports and Methods for Depleting and/or Enriching Library Fragments Prepared from Biosamples |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2023056328A2 true WO2023056328A2 (en) | 2023-04-06 |
WO2023056328A3 WO2023056328A3 (en) | 2023-06-08 |
Family
ID=83995551
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2022/077221 WO2023056328A2 (en) | 2021-09-30 | 2022-09-29 | Solid supports and methods for depleting and/or enriching library fragments prepared from biosamples |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2023056328A2 (en) |
Citations (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1991006678A1 (en) | 1989-10-26 | 1991-05-16 | Sri International | Dna sequencing |
WO2004018497A2 (en) | 2002-08-23 | 2004-03-04 | Solexa Limited | Modified nucleotides for polynucleotide sequencing |
US7057026B2 (en) | 2001-12-04 | 2006-06-06 | Solexa Limited | Labelled nucleotides |
US7115400B1 (en) | 1998-09-30 | 2006-10-03 | Solexa Ltd. | Methods of nucleic acid amplification and sequencing |
US7211414B2 (en) | 2000-12-01 | 2007-05-01 | Visigen Biotechnologies, Inc. | Enzymatic nucleic acid synthesis: compositions and methods for altering monomer incorporation fidelity |
WO2007123744A2 (en) | 2006-03-31 | 2007-11-01 | Solexa, Inc. | Systems and devices for sequence by synthesis analysis |
US7315019B2 (en) | 2004-09-17 | 2008-01-01 | Pacific Biosciences Of California, Inc. | Arrays of optical confinements and uses thereof |
US7329492B2 (en) | 2000-07-07 | 2008-02-12 | Visigen Biotechnologies, Inc. | Methods for real-time single molecule sequence determination |
US20080108082A1 (en) | 2006-10-23 | 2008-05-08 | Pacific Biosciences Of California, Inc. | Polymerase enzymes and reagents for enhanced nucleic acid sequencing |
US7405281B2 (en) | 2005-09-29 | 2008-07-29 | Pacific Biosciences Of California, Inc. | Fluorescent nucleotide analogs and uses therefor |
US7985565B2 (en) | 1997-04-01 | 2011-07-26 | Illumina, Inc. | Method of nucleic acid amplification |
WO2013184796A1 (en) | 2012-06-08 | 2013-12-12 | Illumina, Inc. | Polymer coatings |
WO2014108810A2 (en) | 2013-01-09 | 2014-07-17 | Lumina Cambridge Limited | Sample preparation on a solid support |
US9005891B2 (en) | 2009-11-10 | 2015-04-14 | Genomic Health, Inc. | Methods for depleting RNA from nucleic acid samples |
US9745570B2 (en) | 2009-08-14 | 2017-08-29 | Epicentre Technologies Corporation | Methods, compositions, and kits for generating rRNA-depleted samples or isolating rRNA from samples |
WO2020132304A1 (en) | 2018-12-21 | 2020-06-25 | Epicentre Technologies Corporation | Nuclease-based rna depletion |
WO2021127191A1 (en) | 2019-12-19 | 2021-06-24 | Illumina, Inc. | Designing probes for depleting abundant transcripts |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7361465B2 (en) * | 2004-09-07 | 2008-04-22 | Applera Corporation | Methods and compositions for tailing and amplifying RNA |
US20100331204A1 (en) * | 2009-02-13 | 2010-12-30 | Jeff Jeddeloh | Methods and systems for enrichment of target genomic sequences |
NO2694769T3 (en) * | 2012-03-06 | 2018-03-03 | ||
WO2016081267A1 (en) * | 2014-11-18 | 2016-05-26 | Epicentre Technologies Corporation | Method and compositions for detecting pathogenic organisms |
EP4092136B8 (en) * | 2021-05-20 | 2024-03-13 | Sophia Genetics S.A. | Capture probes and uses thereof |
-
2022
- 2022-09-29 WO PCT/US2022/077221 patent/WO2023056328A2/en active Application Filing
Patent Citations (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1991006678A1 (en) | 1989-10-26 | 1991-05-16 | Sri International | Dna sequencing |
US7985565B2 (en) | 1997-04-01 | 2011-07-26 | Illumina, Inc. | Method of nucleic acid amplification |
US7115400B1 (en) | 1998-09-30 | 2006-10-03 | Solexa Ltd. | Methods of nucleic acid amplification and sequencing |
US7329492B2 (en) | 2000-07-07 | 2008-02-12 | Visigen Biotechnologies, Inc. | Methods for real-time single molecule sequence determination |
US7211414B2 (en) | 2000-12-01 | 2007-05-01 | Visigen Biotechnologies, Inc. | Enzymatic nucleic acid synthesis: compositions and methods for altering monomer incorporation fidelity |
US7057026B2 (en) | 2001-12-04 | 2006-06-06 | Solexa Limited | Labelled nucleotides |
WO2004018497A2 (en) | 2002-08-23 | 2004-03-04 | Solexa Limited | Modified nucleotides for polynucleotide sequencing |
US7315019B2 (en) | 2004-09-17 | 2008-01-01 | Pacific Biosciences Of California, Inc. | Arrays of optical confinements and uses thereof |
US7405281B2 (en) | 2005-09-29 | 2008-07-29 | Pacific Biosciences Of California, Inc. | Fluorescent nucleotide analogs and uses therefor |
WO2007123744A2 (en) | 2006-03-31 | 2007-11-01 | Solexa, Inc. | Systems and devices for sequence by synthesis analysis |
US20080108082A1 (en) | 2006-10-23 | 2008-05-08 | Pacific Biosciences Of California, Inc. | Polymerase enzymes and reagents for enhanced nucleic acid sequencing |
US9745570B2 (en) | 2009-08-14 | 2017-08-29 | Epicentre Technologies Corporation | Methods, compositions, and kits for generating rRNA-depleted samples or isolating rRNA from samples |
US9005891B2 (en) | 2009-11-10 | 2015-04-14 | Genomic Health, Inc. | Methods for depleting RNA from nucleic acid samples |
WO2013184796A1 (en) | 2012-06-08 | 2013-12-12 | Illumina, Inc. | Polymer coatings |
WO2014108810A2 (en) | 2013-01-09 | 2014-07-17 | Lumina Cambridge Limited | Sample preparation on a solid support |
WO2020132304A1 (en) | 2018-12-21 | 2020-06-25 | Epicentre Technologies Corporation | Nuclease-based rna depletion |
WO2021127191A1 (en) | 2019-12-19 | 2021-06-24 | Illumina, Inc. | Designing probes for depleting abundant transcripts |
Non-Patent Citations (31)
Title |
---|
ABU-ALI, G. S. ET AL., NAT. MICROBIOL., vol. 3, no. 3, 2018, pages 356 - 366 |
AUGUST ET AL., J. BIOL. CHEM., vol. 237, 1962, pages 3786 - 3793 |
BENTLEY ET AL., NATURE, vol. 456, 2008, pages 53 - 59 |
CHAMPOUXSCHULTZ, FEBS J, vol. 276, 2009, pages 1506 - 1516 |
CHO ET AL., NAT. REV. GENET., vol. 13, 2012, pages 260 - 70 |
COCK ET AL., NUCLEIC ACIDS RES., vol. 38, no. 6, 2010, pages 1767 - 71 |
CULVINER ET AL., MBIO, vol. 11, no. 2, 2020, pages e00010 - 20 |
DURACKLYNCH, J. EXP. MED., vol. 216, no. 1, 2019, pages 20 - 40 |
FRANZOSA, E. A. ET AL., PROC. NATL. ACAD. SCI. U. S. A., vol. 111, no. 22, 2014, pages E2329 - 38 |
GIANNOUKOS, G. ET AL., GENOME BIOL, vol. 13, no. 3, 2012, pages R23 |
GILBERT, J. A. ET AL., NAT. MED., vol. 24, 2018, pages 392 - 400 |
GU ET AL., GENOME BIOL, vol. 17, 2016, pages 1 - 13 |
HAISER, H. J. ET AL., SCIENCE, vol. 341, no. 6143, 2013, pages 295 - 298 |
HIGGINS ET AL., FORENSIC SCI MED PATHOL, vol. 10, 2014, pages 56 - 61 |
HRDLICKOVA ET AL., WILEY INTERDISCIP. REV. RNA, vol. 8, no. 1, 2017 |
HUANG ET AL., NUCLEIC ACIDS RES., vol. 48, no. 4, 2020, pages E20 |
KOPYLOVA ET AL., BIOINFORMATICS, vol. 28, no. 24, 2012, pages 3211 - 3217 |
LLOYD-PRICE ET AL., GENOME MED, vol. 8, 2016, pages 51 |
LLOYD-PRICE, J. ET AL., NATURE, vol. 569, 2019, pages 655 - 662 |
MODAKSRINIVASAN, J. BIOL. CHEM., vol. 248, no. 19, 1973, pages 6904 - 6910 |
MOHANTYKUSHNER, MOL. MICROBIOL., vol. 34, 1999, pages 1094 - 1108 |
O'HARA ET AL., PROC. NATL. ACAD. SCI. U. S. A., vol. 92, 1995, pages 1807 - 1811 |
PETERSEN ET AL., MICROBIOME, vol. 5, no. 1, 2017, pages 98 |
PREZZA, G. ET AL., RNA, vol. 26, 2020, pages 1069 - 1078 |
QUAST ET AL., NUCLEIC ACIDS RES, vol. 41, 2013, pages D590 - 6 |
RECK, M. ET AL., BMC GENOMICS, vol. 16, no. 1, 2015, pages 494 |
SCHULTZ ET AL., J. BIOL. CHEM., vol. 281, 2006, pages 1943 - 1955 |
STEWART ET AL., NATURE, vol. 562, 2018, pages 583 - 588 |
TURNBAUGH, P. J. ET AL., PROC. NATL. ACAD. SCI. U. S. A., vol. 107, 2010, pages 7503 - 7508 |
TURRONI ET AL., PLOS ONE, vol. 7, no. 5, 2012, pages e36957 |
ZHAO ET AL., SCI. REP., vol. 8, no. 1, 2018, pages 4781 |
Also Published As
Publication number | Publication date |
---|---|
WO2023056328A3 (en) | 2023-06-08 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN113166797B (en) | Nuclease-based RNA depletion | |
RU2708337C2 (en) | Methods and compositions for dna profiling | |
EP2949757B1 (en) | Method for multiplexing amplification reactions | |
EP2576815B1 (en) | Method for the prognosis of colorectal cancer | |
TW201321518A (en) | Method of micro-scale nucleic acid library construction and application thereof | |
EP2376631A1 (en) | Method for analysis of nucleic acid populations | |
EP3177740A1 (en) | Digital measurements from targeted sequencing | |
JP2007502610A (en) | Amplification method | |
JP2002330783A (en) | Concentration and amplification of target for analyzing array | |
EP3538662A1 (en) | Methods of producing amplified double stranded deoxyribonucleic acids and compositions and kits for use therein | |
CN110959045B (en) | Improved methods and kits for generating large-scale parallel sequenced DNA libraries | |
JP2020505045A (en) | Barcoded DNA for long range sequencing | |
EP3607064A1 (en) | Method and kit for targeted enrichment of nucleic acids | |
CN112176058A (en) | Probe library, method and kit for detecting tumor biomarkers | |
WO2011153684A1 (en) | Method and kit for the prognosis of colorectal cancer | |
US20180291436A1 (en) | Nucleic acid capture method and kit | |
WO2018186947A1 (en) | Method and kit for targeted enrichment of nucleic acids | |
EP2689035B1 (en) | Method and kit for determining in vitro the probability for an individual to suffer from colorectal cancer | |
CN113026111A (en) | Kit for constructing human single cell TCR sequencing library and application thereof | |
US20230094911A1 (en) | Solid Supports and Methods for Depleting and/or Enriching Library Fragments Prepared from Biosamples | |
WO2008066979A2 (en) | Methods for rapid, single-step strand displacement amplification of nucleic acids | |
WO2023056328A2 (en) | Solid supports and methods for depleting and/or enriching library fragments prepared from biosamples | |
WO2022007863A1 (en) | Method for rapidly enriching target gene region | |
CA3168563A1 (en) | Parallelized sample processing and library prep | |
CN117545852A (en) | Solid support and method for depleting and/or enriching library fragments prepared from biological samples |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 22794038 Country of ref document: EP Kind code of ref document: A2 |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2022354071 Country of ref document: AU Ref document number: AU2022354071 Country of ref document: AU |
|
WWE | Wipo information: entry into national phase |
Ref document number: 3223045 Country of ref document: CA |
|
ENP | Entry into the national phase |
Ref document number: 2022354071 Country of ref document: AU Date of ref document: 20220929 Kind code of ref document: A |